HORI, Sayaka et al. (2021) International Worm Meeting "Contribution of a FOXD3/4 ortholog to optimization of avoidance behavior mediated by pre- and postsynaptic gene expression for a biphasic calcium response"

Mitani, Shohei, Hori, Sayaka

[International Worm Meeting, 2021]

The central neural network optimizes avoidance behavior depending on the nociceptive stimulation intensity is essential for survival. How the property of the hub neurons for selecting behaviors is genetically defined is not well understood. Here we show that the transcription factor unc-130, a human FOXD3/4 ortholog, is required to optimize the avoidance behavior depending on stimulus strength in Caenorhabditis elegans. unc-130 is necessary for both ON-calcium decrease and OFF-calcium increase in AIB, a central neuron of avoidance optimization. Ablation of predicted upstream inhibitory neurons reduced frequency of turn behavior, suggesting optimization needs both calcium responses. At a molecular level, unc-130 upregulates expression of at least three genes: nca-2, a homolog of the vertebrate cation leak channel NALCN, glr-1, an AMPA type glutamate receptor, and eat-4, a hypothetical L-glutamate transmembrane transporter in the central neuron of optimization. unc-130 shows more limited regulation in optimizing behavior than an atonal homolog lin-32, unc-130 and lin-32 appear to act in parallel molecular pathways. Our findings suggest that unc-130 contributes to behavioral optimization mediated by pre- and postsynaptic expression to lead biphasic neural responses.

O'Brien, Barbara et al. (2015) International Worm Meeting "The EGF-like transmembrane protein c-tomoregulin acts in the UNC-6/Netrin pathway to mediate dendrite self-avoidance ."

O'Brien, Barbara, Smith, Cody, Miller, David, O'Brien, Timothy, Hori, Sayaka, Mitani, Shohei

[International Worm Meeting, 2015]

The dendritic processes of nociceptive neurons transduce external signals into neurochemical cues that alert the organism to noxious stimuli. The receptive field for each sensory neuron is defined by sister dendrites which occupy discrete domains and do not overlap. This phenomenon of dendrite self-avoidance constitutes a fundamental patterning mechanism during neural development. Two neurons in C. elegans, PVDL and PVDR are model nociceptors for studying the development of dendritic self-avoidance because they exhibit an elaborate but well-characterized dendritic arbor that is readily visible. We have shown that the diffusible cue, UNC-6/Netrin, mediates PVD self-avoidance in conjunction with its receptors UNC-40/DCC and UNC-5. An independent genetic screen showed that c-tomoregulin mutants show increased overlap of PVD dendritic branches (5% in wild type vs. 23% in mutants, p<0.001). Restoration of c-tomoregulin to PVD rescued this self-avoidance defect, indicating that expression of c-tomoregulin in PVD is sufficient to mediate self-avoidance. Further, c-tomoregulin tagged with GFP shows localization throughout the dendritic branches of PVD neurons. The mammalian homolog, h-tomoregulin, is highly expressed in the nervous system; similarly, c-tomoregulin is also expressed in neurons in C. elegans. Double mutants of c-tomoregulin with either unc-5 or unc-40 do not show enhanced overlap compared to their single mutant counterparts, suggesting that c-tomoregulin functions in the UNC-6 pathway to mediate self avoidance. The goal of this work is to elucidate the cell biological pathways that regulate self-avoidance and the role of tomoregulin proteins in this evolutionarily conserved process. .

Kage-Nakadai, Eriko et al. (2011) International Worm Meeting "Low-copy integration of transgenes by TMP/UV methods."

Yoshina, Sawako, Gengyo-Ando, Keiko, Kage-Nakadai, Eriko, Kobuna, Hiroyuki, Funatsu, Osamu, Mitani, Shohei, Ootori, Muneyoshi, Hori, Sayaka

[International Worm Meeting, 2011]

In Caenorhabditis elegans, transgenic strains are typically generated by injecting DNA into the germline to form high-copy extrachromosomal arrays. These transgenes are semi-stable and their expression are silenced in germline. In order to create single- or low-copy chromosomal integrated lines, methods using Mos1 transposon or microparticle bombardment have been developed. We have developed an alternative method by using TMP/UV, that produces low-copy integrations. We have successfully integrated low-copy of transgenes using positive selection with vps-45 rescue fragment / temperature sensitivity and negative selection with ben-1 rescue fragment / benomyl sensitivity. We confirmed that low-copy integrants express transgenes in germline. Currently, using this method, we are constructing a PhiC31-attBP / Cre-LoxP system.

Miller-Fleming, Tyne W. et al. (2015) International Worm Meeting "The degenerin/epithelial sodium channel protein UNC-8 drives activity-dependent synaptic remodeling in GABAergic neurons."

Manning, Laura, Miller III, David M., Richmond, Janet E., Hori, Sayaka, Matthewman, Cristina, Gornet, Megan, Petersen, Sarah C., Mitani, Shohei, Bianchi, Laura, Miller-Fleming, Tyne W.

[International Worm Meeting, 2015]

Developing neural circuits undergo extensive refinement mediated by transcriptional mechanisms and environmental cues; however, the specific molecules that mediate these interactions are uncharacterized. The Degenerin/Epithelial Sodium Channel (DEG/ENaC) family of proteins has been shown to play a role in mammalian and invertebrate plasticity and here, we identify the DEG/ENaC protein UNC-8 as a critical regulator of GABAergic synaptic remodeling in C. elegans. We see that UNC-8 is required in GABA neurons for the proper removal of synapses in a transcriptionally controlled and activity-dependent remodeling mechanism. UNC-8 drives GABA neuron remodeling in parallel to the Iroquois family homeodomain transcription factor, IRX-1. Additionally, UNC-8 cation channel function is necessary to dismantle presynaptic domains. We find that UNC-8 functions in a common genetic pathway with neurotransmitter release. Our results suggest that UNC-8 functions as a sensor of GABAergic activity to promote synapse disassembly, thereby serving as a potential link between genetic mechanisms and neurotransmission in the developing nervous system. .

Miller, Tyne W et al. (2013) International Worm Meeting "The degenerin family ion channel UNC-8 promotes activity-dependent remodeling of GABAergic synapses in C. elegans."

Wang, Ying, Miller, Tyne W, Bianchi, Laura, Petersen, Sarah C, Miller, David M, Hori, Sayaka, Richmond, Janet E, Matthewman, Cristina, Gornet, Megan E, Mitani, Shohei, Lu, Han

[International Worm Meeting, 2013]

Synaptic networks are extensively remodeled in the developing brain by mechanisms that require neural activity. Members of the DEG/ENaC (Degenerin/Epithelial sodium channel) family are known to modulate plasticity in the mammalian brain, but the molecular events that regulate this effect are poorly defined. Here we describe an activity-dependent mechanism in which the DEG/ENaC protein, UNC-8, promotes synaptic remodeling in C. elegans. GABAergic Dorsal D (DD) motor neurons reverse polarity by relocating synapses from ventral to dorsal muscles in the first larval stage. This pathway is blocked by the UNC-55/COUP-TF transcription factor in Ventral D (VD) GABAergic neurons, which ectopically remodel in an unc-55 mutant. We exploited this mutant phenotype in a cell-specific profiling strategy to identify UNC-55-regulated transcripts. This approach revealed fifty UNC-55 targets that are required for GABA neuron remodeling, including UNC-8. We find that UNC-8 functions in DD neurons where it is localized near ventral DD synapses. Our results show that UNC-8 is required for removing ventral synapses in a mechanism that depends on GABA. A necessary role for neurotransmitter release is also suggested by our finding that a voltage-gated calcium channel subunit, UNC-2, promotes remodeling. In vitro reconstitution of an UNC-8 channel results in robust cation transport activity that is strongly inhibited by extracellular calcium. The negative effect of calcium on UNC-8 function is consistent with a model in which depletion of extracellular calcium by UNC-2 at active GABAergic synapses effectively relieves the calcium block and thereby induces UNC-8 activation. Our results support a model in which UNC-8 functions as an activity sensor in GABAergic DD motor neurons to trigger the deconstruction of ventral synapses, thus promoting the remodeling process.

Edgley, Mark et al. (2015) International Worm Meeting "Comprehensive Biology: How do we complete the C. elegans Knockout Project."

Moerman, Donald, Suehiro, Yuji, Motohashi, Tomoko, Taylor, Jon, Au, Vinci, Iwata, Satoru, Miller, Angela, Hori, Sayaka, Flibotte, Stephane, Yoshina, Sawako, Fernando, Lisa, Mitani, Shohei, Dejima, Katsufumi, Raymant, Greta, Edgley, Mark

[International Worm Meeting, 2015]

The primary objective of this project is to provide the research community with severe loss-of-function mutations of all C. elegans genes. To date we, and the community, have identified such mutations for over14,000 of the 20,000 genes in the worm (G3 2012 2:1415-25; Genome Res. 2013 23:1749-63). Strains harboring these mutations are available through the Caenorhabditis Genetics Center and all data pertaining to the mutations is available at WormBase.We are using three strategies to generate and isolate mutations in the remaining 6,000 genes. In the first strategy we will continue to isolate deletion mutants using targeted PCR after UV/TMP mutagenesis. This has been a major method for both nodes to identify mutations in non-essential and essential genes until recently, but will now be performed only at the Tokyo node.In the second strategy we are using random mutagenesis coupled to Whole Genome Sequencing (WGS) to identify new mutations, focusing on mutations that occur in the remaining 6,000 genes that do not have null alleles (Vancouver node). This strategy will allow us to identify mutations in both non-essential and essential genes and to recover the mutant animals. Using WGS we have recently identified nonsense and splicing defects in over 2,000 genes, of which about 500 are among the remaining 6,000. We are currently working to isolate each of these mutations as homozygous lines, or as balanced recessive lethal strains. [Also see poster from the Mitani group on UV/TMP/WGS to identify deletions].In the third strategy we are using CRISPR/Cas9 technology to target specific genes as a complement to the random approach described above. We are particularly interested in using this method to target the remaining members of large multi-gene families, for example kinases and transcription factors. We will also use it to expedite community requests if we do not already have hits through the deletion and WGS projects.