Zhou, Xin et al. (2021) International Worm Meeting "The HSPG Syndecan is a core organizer of cholinergic synapses in C. elegans"

Cizeron, Melissa, Zhou, Xin, Romatif, Oceane, Vachon, Camille, Jospin, Maelle, Bessereau, Jean-Louis, Bulow, Hannes E.

[International Worm Meeting, 2021]

The extracellular matrix has emerged as an active component of chemical synapses regulating synaptic formation, maintenance and homeostasis. The heparan sulfate proteoglycan syndecans are known to regulate cellular and axonal migration in the brain. They are also enriched at synapses, but their synaptic functions remain more elusive. Here we show that SDN-1/Syndecan is a key organizer of the neuromuscular junctions (NMJs) and is the core component that clusters the homomeric alpha7-like nicotinic receptor ACR-16 at cholinergic NMJs. SDN-1 is concentrated at neuromuscular junctions (NMJs) by the neurally-secreted synaptic organizer MADD-4/Ce-Punctin. Punctin is secreted by cholinergic and GABAergic motoneurons into the synaptic cleft and triggers appropriate postsynaptic differentiation. We now show that Punctin has at least two parallel functions at cholinergic NMJs. First, it localizes the syndecan produced by muscle cells at postsynaptic sites. Second, it concentrates and likely activates the netrin receptor UNC-40/DCC. Those cooperatively recruit the FRM-3/FARP and LIN-2/CASK through direct interaction with the PDZ domain binding site of SDN-1 and the C-terminal P3 domain of UNC-40, respectively. The FERM-FA domain of FRM-3/FARP also engages direct interaction with SDN-1, likely with its submembrane C1 domain. The resulting CASK/FARP/Syndecan complex localizes N-AChRs at cholinergic NMJs through physical interactions. Interestingly, we were able to relocalize N-AChRs at GABAergic NMJs using a chimera containing the extracellular domain of the NLG-1/Neuroligin and the intracellular domain of SDN-1. Therefore, SDN-1 stands at the core of the cholinergic synapse organization by bridging the extracellular synaptic determinants to the intracellular synaptic scaffold that controls the postsynaptic receptor content. Surprisingly, the molecular mechanisms that control alpha7-like nicotinic receptors localization and dynamics are still largely unknown. Because all the components identified in C. elegans are evolutionarily conserved and expressed in mammalian neurons, our results provide a new framework to test if syndecan regulates the localization of nicotinic receptors in the mammalian brain.

Correa, Edgar et al. (2021) International Worm Meeting "The conserved transcription factor UNC-30/PITX1-3 coordinates synaptogenesis with cell identity in C. elegans GABA motor neurons."

Bessereau, Jean-Louis, Mialon, Morgane, Kratsios, Paschalis, Correa, Edgar, Pinan-Lucarre, Berangere, Zhou, Xin

[International Worm Meeting, 2021]

During nervous system development, it is critical for neurons to establish functional synapses. This process relies on the ability of a presynaptic neuron to synthesize, package, and release a specific neurotransmitter, and the ability of a postsynaptic neuron to present the correct neurotransmitter receptor. However, the molecular mechanisms that coordinate these distinct events, occurring at a pre- and a post-synaptic cell, are poorly understood. The nematode C. elegans represents a powerful model to study synapse development due to its known connectome, powerful genetics, and single-cell resolution analysis. The evolutionarily conserved transcription factor (TF) UNC-30/PITX1-3 has been shown to control neuronal communication between nerve cord GABAergic motor neurons (MNs) and body-wall muscle by directly activating the expression of GABA biosynthesis genes (e.g., unc-25/GAD, unc-46/LAMP, unc-47/VGAT) in the presynaptic side. Our preliminary data shows that animals lacking unc-30 gene activity display defects in GABA-Receptor (GABA-R) clustering in the postsynaptic side, although UNC-30 is not expressed in muscle. Intriguingly, the same GABA-R clustering defect is observed in animals lacking the short isoform of madd-4/Punctin (madd-4S), a secreted synaptic organizer produced by GABA MNs. Thus, we hypothesize that UNC-30 controls the establishment of functional synapses by activating GABA biosynthesis genes and madd-4S. We found that madd-4S expression is reduced in GABA MNs of unc-30 mutant animals. Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) suggests UNC-30 controls madd-4S directly, a notion we confirmed by deleting the UNC-30 binding site in the context of the endogenous madd-4S locus. Besides acting as an activator of GABA synthesis genes and madd-4S, our preliminary data also suggests UNC-30 is required to prevent the adoption of alternative neuronal identities in GABA MNs. We found that several genes normally expressed in cholinergic MNs (e.g., madd-4L, glr-5, unc-53) become ectopically expressed in GABA MNs of unc-30 mutant animals. These findings together with ChIP-Seq data suggest an additional role for UNC-30 as direct repressor of alternative identities. Since these analyses rely on a null unc-30 allele that disrupts gene activity throughout all life stages, ongoing experiments will determine whether UNC-30 is continuously required to maintain proper gene expression in GABA MNs. Altogether, these findings suggest UNC-30/PITX1-3 is a transcriptional link for the coordination of synaptogenesis with cell identity features in C. elegans GABA MNs.

Tao Xu (2008) C.elegans Neuronal Development Meeting "Towards a better understanding of docking/priming of dense core vesicles in C. elegans neurons"

Tao Xu

[C.elegans Neuronal Development Meeting, 2008]

Neuropeptides are packed into dense core vesicles (DCVs) and play critical roles in synaptic signaling. Before a DCV undergoes Ca-triggered exocytosis, it has to become fusion-competent through a so-called docking/priming step. Although a few molecules have been suggested to be involved in the docking/priming of DCVs, our understanding of the mechanism of this process is far from complete. Recently, we have developed high resolution functional assays to monitor the docking/priming process of DCVs in C. elegans neurons (Zhou et al., 2007, Neuron). Combining these techniques with the power of genetic manipulation in C. elegans model systems allows the illustration of sequential events in the docking/priming of DCVs at an unprecedented systematic level. I will summarize our recent progress towards a clearer picture of docking/priming of DCVs through analyzing the C. elegans mutants which are implicated to affect docking/priming.

D Omura et al. (1999) International C. elegans Meeting "A screen for mutations that affect the localization of CED-4"

F Chen, B Horvitz, B Hersh, D Omura

[International C. elegans Meeting, 1999]

In the programmed cell death pathway, ced-4 is a key activator of the C. elegans caspase ced-3 . Genetically, ced-4 is upstream of ced-3 and is necessary for normal programmed cell death to occur. egl-1 and ced-9 act upstream of ced-4 in the genetic pathway and are involved in activating and inhibiting ced-4 activity, respectively. Previous work has shown that the localization of ced-4 seems intimately tied to its activity. In cells that are not undergoing programmed cell death, CED-4 is localized to mitochondrial membrane surfaces. When these cells are induced to die by overexpressing egl-1 or by ced-9 loss-of-function, CED-4 is localized to the perinuclear membrane 1 . CED-4 localization appears to correlate with the life-or-death decision of a cell, but how and why the localization occurs is unknown. When overexpressed, a CED-4::GFP fusion protein appears to localize to the mitochondria and perinuclear region of cells that are not undergoing programmed cell death. To identify factors important for the localization of CED-4, we will screen for mutants in which this CED-4::GFP fusion protein fails to localize to the perinucleus of viable cells. 1 Hersh, B., Chen, F., Conradt, B., Zhou, Z., and Horvitz, HR (1999) 12th International C. elegans Meeting

Frederick J Tan et al. (2004) West Coast Worm Meeting "Functions of the Outer Mitochondrial Membrane Protein CED-9"

R Blake Hill, Andrew Z Fire, Frederick J Tan

[West Coast Worm Meeting, 2004]

CED-9 (CEll Death abnormal) negatively regulates the programmed cell death pathway in C. elegans . This membrane associated pro-survival protein prevents the activation of the CED-3 caspase. Loss of function ced-9 animals arrest at early embryogenesis, presumably dying from ectopic cell deaths. Conversely, gain of function animals appear healthy, albeit with extra cells (Hengartner, Ellis and Horvitz, 1992). These effects have been ascribed to the ability of CED-9 to sequester the CED-4/CED-3 caspase complex at the mitochondria (Chen, et al. , 2000). We hope to understand the structural features of CED-9 that participate in its function. To this end, we are assaying the activity of various CED-9 mutants and derivatives for ability to prevent cell death and respond to appropriate modulating signals. A question that we consider to be central is whether the C-terminal transmembrane domain of CED-9 serves only as a targeting sequence to the mitochondrion, or plays a more active role. We have demonstrated that this domain is essential for mitochondrial localization and plays some role in the function of CED-9. Distinction between localization and other structural and functional roles is in progress. Hengartner MO, Ellis RE and HR Horvitz. 1992. Caenorhabditis elegans gene ced-9 protects cells from programmed cell death. Nature 356 , 494-499. Chen F, Hersh BM, Conradt B, Zhou Z, Riemer D, Gruenbaum Y, and HR Horvitz. 2000. Translocation of C. elegans CED-4 to Nuclear Membranes During Programmed Cell Death. Science 287 , 1485-1489.

S Chen et al. (1999) International C. elegans Meeting "Are Complex N-glycans Essential for the Development of the Nematode C. elegans?"

AM Spence, S Zhou, S Chen, M Sarkar, H Schachter

[International C. elegans Meeting, 1999]

UDP-N-acetylglucosamine:alpha-3-D-mannoside beta-1,2-N-acetylglucosaminyltransferase I (GnT I) is a key enzyme in the synthesis of hybrid and complex N-glycans. Disruption of the GnT I gene in the mouse results in arrest of embryogenesis. Defects in the synthesis of complex N-glycans have been shown to be associated with several human congenital diseases. We are investigating the role of complex N-glycans in C. elegans . Three C. elegans genes homologous to mammalian GnT I have been identified by searching the genome databases. They are designated gly-12, gly-13 and gly-14 . cDNAs encoding these predicted genes have been cloned and their expression patterns determined. Both gly-12 and gly-14 encode active enzymes while gly-13 does not, towards the substrate tested [Chen et al ., 1999]. To better understand its biological function, we wanted to isolate a GnT I null mutant. We screened worms that were UV irradiated in the presence of trimethylpsoralen(TMP) and isolated a mutant with a 1.6kb deletion of gly-12 . The deletion covers from intron 6 to exon 12, a DNA fragment encoding most of the GnT I C-terminal catalytic domain. The mutants have no obvious phenotypic defects. A Tc1 insertion has been detected within intron 9 of gly-14 . Screening for Tc1 derived deletions is under way. We will create double mutants that lack both gly-12 and gly-14 to test the redundancy of these two GnT I genes. Chen, S., Zhou, S., Sarkar, M., Spence, A. M., and Schachter, H. (1999) J. Biol. Chem. 274 , 288-297.

Kim Reese et al. (2001) International C. elegans Meeting "A novel protein required for degradation of CCCH finger proteins in somatic lineages."

Geraldine Seydoux, Kim Reese

[International C. elegans Meeting, 2001]

The first cleavages of the embryo are asymmetric and give rise to 5 somatic lineages and the embryonic germ lineage. Several maternal proteins are asymmetrically segregated during these cleavages: in particular three proteins, PIE-1, POS-1 and MEX-1, segregate preferentially with the germ lineage and are excluded from somatic lineages. These proteins have in common a pair of CCCH zinc fingers (ZF1 and ZF2). We have found that, when fused to GFP, ZF1s from PIE-1, MEX-1 and POS-1 cause GFP to be degraded specifically in somatic blastomeres. Consistent with a role in protein degradation, mutations in PIE-1 ZF1 cause abnormal stabilization of PIE-1 in somatic blastomeres. These observations suggest that the ZF1 in PIE-1, MEX-1 and POS-1 targets these proteins for degradation specifically in somatic blastomeres. To identify factors that function in this process, we performed a yeast-two hybrid screen for proteins that interact with PIE-1 ZF1 and identified a novel protein, F59B2.6 (thanks to Zheng Zhou and Bob Horvitz for their excellent library). F59B2.6 does not appear to have any recognizable motifs. GST pull down experiments have confirmed that F59B2.6 interacts directly and specifically with ZF1 and does not interact with ZF2. RNA-mediated interference of F59B2.6 causes abnormal accumulation of PIE-1,MEX-1 and POS-1 in somatic blastomeres, and results in embryonic lethality. F59B2.6(RNAi) embryos show no defects in PAR-2, PAR-6 and GLP-1 localization, indicating that this gene is not generally required for embryonic polarity. These observations suggest that F59B2.6 recognizes ZF1-containing proteins and targets them for degradation in somatic blastomeres. Although CCCH fingers in other proteins (e.g. mammalian TIS11) have been implicated in binding to RNA, our findings raise the possibility that CCCH fingers can also function as determinants of protein stability.

Sarah C. Anthony et al. (2007) International Worm Meeting "Microarray profiling of C. elegans GABAergic motor neurons to reveal synaptic remodeling genes."

Sarah C. Anthony, Stephen E. Von Stetina, Joseph D. Watson, David M. Miller III, Bill Walthall

[International Worm Meeting, 2007]

Neurons adopt polarized morphologies to set the direction of information flow in the nervous system. Neurons in diverse species are universally capable of altering axonal versus dendritic compartments in response to injury or through developmentally regulated programs. One striking example of this phenomenon is the remodeling of GABAergic motor neurons in C. elegans. Dorsal D or DD motor neurons initially innervate ventral muscle but switch polarity after hatching to synapse with dorsal muscle. This polarity switch occurs as ventral D (VD) motor neurons arise in L1 larvae to innervate ventral muscles. In unc-55 mutants, VD motor neurons adopt the dorsal polarity of DD motor neurons thereby resulting in simultaneous innervation (inhibition) of dorsal muscles by both DD and VD motor neurons. UNC-55, a member of the COUP family of nuclear hormone receptors, is expressed specifically in VD motor neurons. Thus, UNC-55 appears to function as a transcriptional switch that prevents VD motor neurons from adopting the dorsal polarity of DD motor neurons (Zhou and Walthall, 1998). Our goal is to identify UNC-55 regulated genes that govern D motor neuron synaptic remodeling. For this purpose, we are using the mRNA tagging method to generate microarray profiles of GABAergic neurons. We used the D motor neuron-specific promoter, C04G2.1, to drive expression of epitope-tagged poly-A-Binding Protein (3X FLAG-PAB-1) in DD and VD motor neurons. An mRNA tagging profile of wildtype L2 larvae using this strain detects established GABAergic and neuronal transcripts including unc-55. With this reference profile of normal D-type motor neurons, we can now identify dynamically regulated genes in unc-55 gain-of-function and loss-of-function mutants. This strategy is expected to reveal UNC-55 target genes with key roles in motor neuron synaptic remodeling.

Yingsong Hao et al. (2001) International C. elegans Meeting "USING YEAST-TWO HYBRID AND RNA-MEDIATED INTERFERENCE TO IDENTIFY PROTEINS ESSENTIAL FOR PIE-1 ACTIVITY AND LOCALIZATION IN EARLY EMBRYOS"

Geraldine Seydoux, Yingsong Hao

[International C. elegans Meeting, 2001]

The CCCH zinc finger protein PIE-1 is an essential regulator of germ cell fate that segregates with the germ lineage in early C. elegans embryos. PIE-1 has at least two functions in the embryonic germ lineage: 1) inhibition of mRNA transcription, and 2) promotion of efficient NOS-2 expression, a maternal protein required for primordial germ cell development. Structure-function studies have indicated that these two functions require sequences in the C-terminal region of PIE-1 (Tenenhaus et al., 2001). The C-terminus of PIE-1 is also required for asymmetric segregation of PIE-1 in dividing germline blastomeres (Reese et al., 2000). We have used the C-terminus of PIE-1 as a bait in a yeast two hybrid screen in the hope of identifying proteins required for PIE-1 activity and/or localization. Screening of 900,000 transformants yielded 365 candidate interactors (thanks to Zheng Zhou and Bob Horvitz for their excellent library). To quickly identify functionally relevant candidates, we designed a simple protocol to test the in vivo function of each candidate by RNAi. We first isolate the "prey" plasmids from the positive yeast transformants by selection on an auxotrophic E. coli strain. The prey inserts are then PCR amplified and transferred by recombinational cloning into Lisa Timmons and Andy Fire’s feeding vector and transformed into E. coli strain HT115. Resulting transformants are used to "feed" three tester worm strains: 1) a MED-1:GFP line (thanks to Morris Maduro and Joel Rothman) to assay for defects in transcriptional repression, 2) a GFP: nos-2 3’UTR line to assay for defects in NOS-2 expression, and 3) a PIE-1:GFP line to assay for defects in PIE-1 localization. The screen is ongoing and progress will be reported at the meeting.

DEVKOTA, RANJAN et al. (2015) International Worm Meeting "Novel regulators of the C. elegans UNC-6/Netrin pathway."

DEVKOTA, RANJAN, Garriga, Gian, Morck, Catarina, CHIEN, JASON

[International Worm Meeting, 2015]

Precise targeting of axons is essential for the proper functioning of the nervous system. Axonal growth cones respond to various guidance cues and navigate through a complex environment to reach their synaptic targets1. Even though many guidance cues and receptors have been identified, there is less knowledge of how axons respond to different cues in different phases of their growth. The aim of this project is to enhance the understanding of axon guidance by focusing on the development of the bilaterally symmetric Hermaphrodite Specific Neurons (HSNs). Each HSN extends a single axon to the ventral nerve cord, which then turns anteriorly and extends to the nerve ring2. The HSNs innervate vulval muscles and the AVF neurons to stimulate hermaphrodite egg laying and locomotion, respectively3,4. We have isolated three mutants whose HSN axons fail to extend ventrally. Our preliminary mapping, complementation and genetic interaction data suggest that these mutations define three genes that function together to antagonize the conserved UNC-6/Netrin signaling pathway. Our initial goal is to identify the underlying mutations with whole genome sequencing and rescue experiments. The identification of these genes will shed light on how the UNC-6/Netrin pathway is modulated for proper axon guidance along the dorsal-ventral axis.References:1. Dickson, B. J. Molecular mechanisms of axon guidance. Science 298, 1959-1964(2002).2. Garriga, G., Desai, C. & Horvitz, H. R. Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Caenorhabditis elegans. Development 117, 1071-1087(1993).3. Desai, C., Garriga, G., McIntire, S. L. & Horvitz, H. R. A genetic pathway for the development of the Caenorhabditis elegans HSN motor neurons. Nature 336, 638-646(1988).4. Hardaker, L. A., Singer, E., Kerr, R., Zhou, G. & Schafer, W. R. Serotonin modulates locomotory behavior and coordinates egg-laying and movement in Caenorhabditis elegans. J. Neurobiol. 49, 303-313(2001). .