Cherkasova VA et al. (1993) International C. elegans Meeting "The C. elegans dauer larva as a nonaging developmental variant:the study of dauer-overexpressed genes during development and aging."

Shmookler Reis RJ, Kurz B, Egilmez NK, Cherkasova VA

[International C. elegans Meeting, 1993]

Laura B Metz et al. (2001) International C. elegans Meeting "egl-2(lf) Mutants are Volatile Anesthetic Resistant"

C Michael Crowder, Laura B Metz, Bruno van Swinderen

[International C. elegans Meeting, 2001]

In screening through new and existing mutants for resistance to volatile anesthetics (VAs), strains carrying egl-2(rf) mutations were found to be VA resistant. egl-2 codes for a homolog of the Drosophila ether-a-go-go potassium channel ( eag ). Drosophila eag(lf) mutants were originally isolated based on their convulsion-like phenotype in the presence of the VA diethyl ether. Of the various mutants that we have found to be abnormally sensitive to VAs, egl-2(rf) mutants are unique in that they do not appear to have alterations in synaptic transmitter release. Thus, we hypothesized that egl-2 may regulate VA sensitivity through a mechanism distinct from the goa-1, egl-30, unc-64 mechanism(s), all of which regulate both VA sensitivity and synaptic transmitter release. We constructed double mutants containing egl-2(null) and either goa-1(null) (VA resistant) or unc-64(js21) (VA hypersensitive) or unc-64(md130) (VA resistant). In the case of the unc-64 doubles, the VA phenotypes of double mutants were not significantly different from the unc-64 single mutants. However, the resistance of the goa-1(null);egl-2(null) strain was significantly greater than goa-1(null) and similar to that of egl-2(null). These results suggest that egl-2 functions upstream of unc-64 and downstream or parallel to goa-1 to regulate VA sensitivity. We are currently measuring the VA sensitivity of non-null egl-2 alleles to understand better the sequence requirements for the VA resistance of egl-2(rf) . We are also attempting transformation rescue of the VA resistance of egl-2(lf) with cosmids spanning the egl-2 gene.

Palumbos, Sierra et al. (2021) International Worm Meeting "cAMP controls a trafficking mechanism that directs the neuron specificity and subcellular placement of electrical synapses"

Von Stetina, Steve, Palumbos, Sierra, McWhirter, Rebecca, Miller III, David M, Skelton, Rachel

[International Worm Meeting, 2021]

Despite the functional importance of electrical synapses, the molecular mechanisms that direct the formation of neuron-specific gap junctions remain largely unknown. To address this question, we identified targets of the UNC-4 transcription factor that controls connectivity in the C. elegans motor circuit. UNC-4 functions in VA motor neurons to direct the formation of gap junctions on the VA axon with the interneuron AVA (VA-AVA). Locomotion is disrupted in unc-4 mutants because VAs are miswired with electrical input from the interneuron AVB (VA-AVB) which aberrantly form on the VA soma. Thus, UNC-4 controls both the specificity and subcellular placement of electrical synapses. We determined that UNC-4 blocks expression of two antagonists of cAMP, the phosphodiesterase, PDE-1, and the Go/Gi-coupled GPCR, FRPR-17, to prevent assembly of ectopic VA-AVB gap junctions. This finding suggests that cAMP signaling promotes the formation of functional wild-type VA-AVA gap junctions. We validated this hypothesis by showing that optogenetic elevation of cAMP rescues the Unc-4 movement defect and thus restores VA-AVA circuit function. In addition, forced depletion of cAMP in VAs phenocopies unc-4 mutants. Because gap junction placement is shifted from the VA axon to cell soma in unc-4 mutants, we reasoned that trafficking of gap junction components could be perturbed. Live-cell imaging of the gap junction protein, GFP-UNC-9, confirmed that trafficking into the VA axon is strikingly impaired in unc-4 mutants. Genetic activation of cAMP signaling is sufficient to restore GFP-UNC-9 trafficking in VAs. Thus, we propose that cAMP directs both the specificity and placement of electrical synapses by activating mechanisms that transport gap junction components into the VA axonal compartment. Although studies in cultured cells have implicated cAMP in gap junction assembly, our in vivo experiments now firmly establish that cAMP regulates the biogenesis of electrical synapses in an intact nervous system.

Ammar Hawasli et al. (2001) International C. elegans Meeting "Gain-of-function mutations in egl-30 produce resistance to volatile anesthetics"

Mike Nonet, C Michael Crowder, Stephen Hunt, Owais Saifee, Ammar Hawasli

[International C. elegans Meeting, 2001]

Because mutations in the neuronal syntaxin gene unc-64 alter volatile anesthetic (VA) sensitivity, we have screened for unc-64(rf) suppressors in hopes of identifying regulators of VA action. For different reasons, Owais Saifee in Mike Nonet's lab performed a similar screen. A total of 38,000 genomes were screened by the two labs, and 21 suppressor mutations were isolated. The suppressors were outcrossed from unc-64(rf) and fell into two phenotypic classes loopy and jerky that cosegregate with unc-64(rf) suppression. Two semidominant loopy suppressors mapped by their behavioral and VA resistant phenotypes to chromosome IL near egl-30. egl-30 was sequenced in the mutants, and one of the alleles js126 was found to have a missense mutation in the coding sequence. We did not find an egl-30 mutation in the other strain. egl-30(tg26gf) , kindly given to us by K. Iwasaki, was also loopy and resistant to VAs. Testing of strains transformed with a constitutively active egl-30 array (thanks to Carol Bastiani - Sternberg lab) were also resistant to VAs confirming that egl-30(gf) could indeed produce VA resistance. Furthermore, we treated N2 with phorbol-ester, which activates diacyl glycerol-binding proteins and effectively enhances EGL-30 signaling; treated worms were resistant to VAs. Finally, we found that egl-30(js126) , egl-30(tg26) , the egl-30(gf) array-containing strains, and phorbol ester treated strains were all aldicarb hypersensitive. Aldicarb hypersensitivity suggests that presynaptic release of acetylcholine is increased. Thus, consistent with our previous results for goa-1(rf) and unc-64(rf) that suggested a presynaptic mechanism of VA action, egl-30 regulates VA sensitivity, presumably by controlling transmitter release. Currently, we are constructing double mutants between various VA resistant and hypersensitive strains to place egl-30 in the VA sensitivity pathway.

Stephen E Von Stetina et al. (2002) West Coast Worm Meeting "In search of UNC-4 response elements (U4REs)"

Nina Xu, Penelope J Brockie, David M Miller III, Stephen E Von Stetina, Andres V Maricq

[West Coast Worm Meeting, 2002]

Mutations in the UNC-4 homeodomain transcription factor disrupt backward locomotion. In unc-4 mutants, VA motor neurons fail to receive synapses from their usual interneuron partners and instead accept inputs normally reserved for their lineal sisters, the VB motor neurons. Work done in this laboratory has shown that UNC-4 and the Groucho homolog UNC-37 function together in VA motor neurons to repress VB-specific genes. De-repression of these VB genes in unc-4 mutant VA motor neurons presumptively imposes VB-type inputs. GFP reporters constructed from two genes, del-1 (DEG/ENaC sodium channel subunit) and acr-5 (nicotinic acetylcholine receptor subunit), are ectopically expressed in the VA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins and ion channel components, ACR-5 and DEL-1 are attractive candidates for mediators of neuron-specific synapses.

Palumbos, S.D. et al. (2017) International Worm Meeting "Parallel acting signaling pathways regulate gap junction specificity in the C. elegans motor circuit."

Palumbos, S.D., Skelton, R., Von Stetina, S.E., McWhirter, R., Miller, D.M.

[International Worm Meeting, 2017]

Gap junctions provide the only known form of electrical communication between neurons. Although much has been learned about gap junction assembly, the mechanisms that direct the formation of these "electrical synapses" between specific neurons are largely unknown. Here we address this question in a study to identify transcriptionally-regulated components that control the neuron specificity of gap junctions in the C. elegans motor circuit. VA and VB class motor neurons normally establish electrical synapses with different sets of presynaptic interneurons to drive either backward (VA) or forward (VB) locomotion. The UNC-4 transcription factor is expressed in VA neurons to prevent the adoption of VB-type inputs. unc-4 mutants are unable to crawl backward because VA neurons are miswired with gap junctions from interneurons (AVB) normally reserved for VBs. Work in the Miller lab has shown that UNC-4 preserves functional VA inputs by antagonizing two independent signaling pathways. UNC-4 normally blocks EGL-20/Wnt signaling as well as a parallel acting G-protein-dependent pathway involving GOA-1/GaO to prevent the formation of VB-type connections. In both cases, however, the UNC-4 targets that effectively regulate these downstream pathways are not known. To address this question, we are using a new cell-specific profiling strategy, SeqCel (RNA-Seq of C. elegans cells), to identify unc-4-regulated transcripts in VA neurons. We hypothesize that UNC-4 acts as a transcriptional repressor, and thus predict that unc-4 regulated targets should be over-expressed in unc-4 mutant vs WT VA neurons. Candidate unc-4 targets will be screened by RNAi for suppression of the Unc-4 phenotype (e.g., ectopic gap junctions with AVB). This strategy will likely reveal new regulators of gap junction specificity. Thus, this project applies a quantitative RNAseq profiling strategy to elucidate a fundamental question in developmental neuroscience.

JM Ross et al. (1999) International C. elegans Meeting "UNC-4 and UNC-37 repress VB-specifying genes to define VA traits"

JM Ross, E German, DM Miller, DH Hall

[International C. elegans Meeting, 1999]

VA and VB motor neurons arise as sister cells, but adopt different morphologies and patterns of synaptic input. VA-type synaptic inputs define a circuit for backward movement, while VB-type synaptic inputs define a circuit for forward movement. The UNC-4 homeoprotein is required to specify the VA pattern of synaptic input; in unc-4 mutants VAs receive the VB pattern of synaptic input. We have proposed a model in which UNC-4 acts in the VAs to repress genes that normally specify VB-type inputs (Winnier et al., this meeting). Two observations support the hypothesis that UNC-4 acts as a repressor: 1) UNC-4 requires the activity of UNC-37, a homolog of the Drosophila co-repressor Groucho. 2) The UNC-4 C-terminus contains an Engrailed repressor domain (eh1). We have identified two targets of UNC-4/UNC-37 repression. del-1::gfp is normally expressed in VB motor neurons, but is ectopically expressed in VA motor neurons in unc-4 and unc-37 mutants. acr-5::gfp is normally expressed in VB and DB motor neurons, but is ectopically expressed in VA and DA motor neurons in unc-4 and unc-37 mutants. As cell surface proteins, either DEL-1 (degenerin-like Na channel subunit) or ACR-5 (nicotinic acetylcholine receptor alpha subunit), could specify VB synaptic inputs or define other VB traits. If our model of negative regulation by UNC-4 and UNC-37 is correct, then ectopic expression of UNC-4 in the VBs should repress VB-specific gene expression, and may be sufficient to respecify the VBs with VA-type inputs. To test this hypothesis, we expressed UNC-4 in the VBs using the del-1 promoter. Ectopic expression of UNC-4 in the VBs represses acr-5::gfp , while del-1::gfp expression remains unchanged. Worms harboring an integrated del-1::unc-4 array ( wdIs2 ) display a phenotype which is consistent with the VBs receiving VA-type synaptic inputs and functioning in backward movement: wdIs2 worms are impaired in forward movement and coil ventrally when backing. EM reconstruction of the wdIs2 ventral nerve cord will determine whether this wdIs2 Unc phenotype is a result of miswiring of the VBs with VA-type inputs.

Crowder CM et al. (2000) West Coast Worm Meeting "In vivo characterization of the effects of the unc-64(md130) mutation on anesthetic sensitivity."

Hunt SJ, Crowder CM

[West Coast Worm Meeting, 2000]

We have previously shown that mutations in the neuronal syntaxin gene unc-64 profoundly alter the volatile anesthetic (VA) sensitivity of C. elegans. Two hypomorphic unc-64 alleles confer hypersensitivity to the VAs isoflurane and halothane but a third unc-64 hypomorph is VA resistant. The difference between the isoflurane EC50s (the concentration where the effect is half maximal) of the hypersensitive and resistant alleles is over 30-fold. The high-level resistance produced by the unc-64(md130) mutation indicates that VAs act specifically through a single major mechanism to disrupt coordinated locomotion in C. elegans. The md130 mutation is a single base change at the splice-donor site of the sixth intron, causing some aberrant splicing. By RT-PCR, md130 mutants produce truncated RNAs, in addition to wild-type unc-64 mRNAs. The predicted truncated forms of syntaxin are believed nonfunctional as they lack the transmembrane and part of the H3 domains proven vital for syntaxin's ability to function in neurotransmitter release. The behavioral phenotype of md130 is recessive and similar to other weak reduction-of-function unc-64 alleles. However, the VA-resistance phenotype of md130 is semidominant and behaves as a gain-of-function mutation, consistent with the truncated forms acting dominantly to block VA action. The current studies attempt to address in vivo the molecular mechanism(s) by which the md130 mutation confers this resistance. A plasmid was constructed containing the full-length genomic unc-64(md130) sequence and used to transform wild-type and unc-64(null) animals. These animals are significantly VA resistant. Next we addressed if a truncated product alone could produce the VA phenotype. We constructed a plasmid to produce solely truncated and not wild-type unc-64 product by introducing mutations at both the donor and acceptor splice consensus sequence of the sixth intron. This plasmid was unable to rescue null syntaxin mutants, consistent with no wild-type product being produced; however it successfully knocked-in VA resistance in both the wild-type and null/wild-type backgrounds. Additional plasmids for expressing various syntaxin fragments are currently being constructed to define the specific region(s) of syntaxin that are regulating VA action.

Stephen E. Von Stetina et al. (2007) International Worm Meeting "UNC-4 Represses CEH-12/HB9 to specify synaptic inputs to VA motor neurons in C. elegans."

Rebecca M. Fox, Stephen E. Von Stetina, David M. Miller III, Todd A. Starich, Kathie L. Watkins, Jocelyn E. Shaw

[International Worm Meeting, 2007]

Although the choice of a synaptic partner is an important step in developing a functional nervous system, this process is poorly understood. We use the C. elegans motor circuit as a model to study synaptic specificity. VA and VB motor neurons arise from a common progenitor but accept inputs from different interneurons. The UNC-4 homeodomain protein functions in VA motor neurons with the transcriptional co-repressor, UNC-37/Groucho, to block the creation of chemical synapses with PVC and gap junctions with AVB, interneurons normally reserved for VB sisters. To identify all UNC-4 regulated genes, we adopted a microarray-based approach (mRNA-tagging) in which VA-specific transcripts from wildtype and unc-37 mutant VA motor neurons were hybridized to the C. elegans Affymetrix array (Von Stetina, Fox, et al 2007). ~250 genes show elevated expression in the unc-37 mutant. One of these genes, ceh-12, is homologous to the HB9 homeodomain protein, a well-established determinant of motor neuron fate in flies, birds, and mammals. We have now shown that ceh-12 is a VB specific gene that is normally repressed by unc-4 in VA motor neurons. Transgenic animals in which CEH-12 is ectopically driven in VAs using the unc-4 promoter phenocopy the Unc-4 backward movement defect. These data suggest that CEH-12 is sufficient to miswire VA motor neurons. To test for necessity, we crossed ceh-12 deletion alleles (from M. Edgley and S. Mitani) into unc-4 mutants. Interestingly, ceh-12(0) fully suppresses (e.g. restores backward locomotion) unc-4 hypomorphic mutants but only partially suppresses unc-4 null alleles. These data are consistent with a model in which UNC-4 controls multiple genes to regulate wiring. To determine the effect ceh-12 has on wiring, we visualized electrical synapses between AVB and its motor neuron partners using the labeled gap junction protein UNC-7S::GFP. We observed reduced UNC-7S::GFP puncta on posterior VA motor neurons in ceh-12; unc-4 double mutants (compared to unc-4 mutants alone). These results corroborate the finding that ectopic expression of ceh-12::GFP is confined to posterior VA motor neurons in unc-4 mutants. We conclude that UNC-4 controls at least two partially redundant pathways that function along the anterior-posterior axis to specify inputs to VA motor neurons. We are now conducting RNAi experiments on genes identified by our microarray analysis to reveal other UNC-4 target genes that control synaptic choice in this circuit.

Stephen E Von Stetina et al. (2002) West Coast Worm Meeting "In search of UNC-4 targets."

Stuart K Kim, David M Miller III, Stephen E Von Stetina, Peter J Roy

[West Coast Worm Meeting, 2002]

Mutations in the UNC-4 homeodomain transcription factor disrupt backward locomotion. In unc-4 mutants, VA motor neurons fail to receive synapses from their usual interneuron partners and instead accept inputs normally reserved for their lineal sisters, the VB motor neurons. Work done in this laboratory has shown that UNC-4 and the Groucho homolog UNC-37 function together in VA motor neurons to repress VB-specific genes. GFP reporters constructed from three genes, del-1 (DEG/ENaC sodium channel subunit), acr-5 (nicotinic acetylcholine receptor subunit), and glr-41 (non-NMDA glutamate receptor subunit) are ectopically expressed in the VA motor neurons in unc-4 and unc-37 mutants. We have now performed genetic experiments, however, that rule out a necessary role for either ACR-5 or DEL-1 in the specification of VB-type inputs in both VA and VB neurons. The target genes that are responsible for synaptic choice have remained elusive, despite extensive genetic screens. We will now utilize a whole genome approach to identify the full complement of UNC-4 target genes and test those targets for a role in synaptogenesis.