Open Access Research article

Left-right olfactory asymmetry results from antagonistic functions of voltage-activated calcium channels and the Raw repeat protein OLRN-1 in C. elegans

Sarah L Bauer Huang12, Yasunori Saheki1, Miri K VanHoven12, Ichiro Torayama3, Takeshi Ishihara3, Isao Katsura3, Alexander van der Linden4, Piali Sengupta4 and Cornelia I Bargmann1*

Author Affiliations

1 Howard Hughes Medical Institute and Rockefeller University, New York, NY 10065, USA

2 Herbert W Boyer Program in Biological Sciences, The University of California, San Francisco, San Francisco, CA 94143, USA

3 Structural Biology Center, National Institute of Genetics, and Department of Genetics, The Graduate University for Advanced Studies, Mishima, 411-8540, Japan

4 Department of Biology and National Center for Behavioral Genomics, Brandeis University, Waltham, MA 02454, USA

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Neural Development 2007, 2:24  doi:10.1186/1749-8104-2-24


The electronic version of this article is the complete one and can be found online at: http://www.neuraldevelopment.com/content/2/1/24


Received:23 July 2007
Accepted:6 November 2007
Published:6 November 2007

© 2007 Bauer Huang et al; licensee BioMed Central Ltd.

This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Background

The left and right AWC olfactory neurons in Caenorhabditis elegans differ in their functions and in their expression of chemosensory receptor genes; in each animal, one AWC randomly takes on one identity, designated AWCOFF, and the contralateral AWC becomes AWCON. Signaling between AWC neurons induces left-right asymmetry through a gap junction network and a claudin-related protein, which inhibit a calcium-regulated MAP kinase pathway in the neuron that becomes AWCON.

Results

We show here that the asymmetry gene olrn-1 acts downstream of the gap junction and claudin genes to inhibit the calcium-MAP kinase pathway in AWCON. OLRN-1, a protein with potential membrane-association domains, is related to the Drosophila Raw protein, a negative regulator of JNK mitogen-activated protein (MAP) kinase signaling. olrn-1 opposes the action of two voltage-activated calcium channel homologs, unc-2 (CaV2) and egl-19 (CaV1), which act together to stimulate the calcium/calmodulin-dependent kinase CaMKII and the MAP kinase pathway. Calcium channel activity is essential in AWCOFF, and the two AWC neurons coordinate left-right asymmetry using signals from the calcium channels and signals from olrn-1.

Conclusion

olrn-1 and voltage-activated calcium channels are mediators and targets of AWC signaling that act at the transition between a multicellular signaling network and cell-autonomous execution of the decision. We suggest that the asymmetry decision in AWC results from the intercellular coupling of voltage-regulated channels, whose cross-regulation generates distinct calcium signals in the left and right AWC neurons. The interpretation of these signals by the kinase cascade initiates the sustained difference between the two cells.

Background

Olfactory neurons sense environmental chemicals using large families of chemoreceptor genes that are deployed in elaborate patterns. For example, the main olfactory organs of the nematode Caenorhabditis elegans are the bilateral (left and right) amphids, which house 12 pairs of ciliated sensory neurons. Each sensory neuron expresses many receptor genes, in contrast with vertebrate olfactory neurons that generally express one receptor gene per cell [1]. Every neuron pair expresses a unique complement of receptors, and in addition, expression of some receptor genes is asymmetric on the left and right sides. For example, the left and right ASE gustatory neurons (ASEL and ASER) are structurally similar, but they express different receptor genes and sense different tastants [2,3]. ASE asymmetry, which is established by transcription factors and microRNAs, is stereotyped and tightly coupled to the body plan [4]. The left and right AWC olfactory neurons also have distinct functions, but AWC asymmetry is variable [5]. The receptor gene str-2 is expressed stochastically in one of the AWC neurons, such that half of the animals express str-2 in AWCL and half of the animals express str-2 in AWCR. The AWC cell that expresses str-2 is designated AWCON, and the cell lacking str-2 expression is designated AWCOFF. These alternative AWC gene expression patterns correlate with different olfactory functions: both AWCs sense the odors benzaldehyde and isoamyl alcohol, but only AWCON senses butanone, and only AWCOFF senses 2,3-pentanedione [6]. Behavioral analysis of animals with an altered complement of AWCs has demonstrated that AWC asymmetry increases olfactory discrimination and olfactory plasticity [6,7].

Signaling between the two AWCs is required for the diversification of AWCON and AWCOFF. If one AWC precursor is killed in the embryo, the surviving cell always becomes AWCOFF, suggesting that AWCOFF is a ground state and AWCON is an induced state [5]. The random left-right specification and signaling between equipotential AWCs are reminiscent of developmental lateral signaling, which is usually mediated by Notch receptors and Delta/Serrate ligands [8], but Notch pathway genes have no apparent role in AWC asymmetry [5]. Instead, the induction of AWCON requires NSY-5, an innexin gap junction protein, and NSY-4, a protein similar to claudins and the regulatory γ subunits of voltage-activated calcium channels [9,10]. NSY-5 creates a transient gap junction network essential for communication between the left and right AWCs. NSY-5-dependent ultrastructural gap junctions link the cell bodies of the embryonic AWC neurons with many additional neurons; these gap junctions disappear soon after hatching [9]. Genetic experiments indicate that AWCON induction involves contributions from both the left and right AWC neurons as well as other neurons in the network. nsy-4, which is related to proteins that regulate channels and cell adhesion, also has network functions – it has cell-autonomous effects within the AWC neuron that expresses it, and cell non-autonomous effects on the contralateral AWC [10]. Even in a wild-type genetic background, the level of nsy-4 or nsy-5 activity in one AWC neuron is sensed by the contralateral AWC, so that the neuron with higher nsy-4 or nsy-5 expression preferentially becomes AWCON. It is likely that the networks on the left and right are linked in the nerve ring, where axons from the left and right sides meet [5].

nsy-5 and nsy-4 induce AWCON by repressing a kinase cascade that includes the calcium/calmodulin-dependent kinase II (CaMKII) UNC-43, the p38/JNK mitogen-activated protein kinase kinase kinase (MAPKKK) NSY-1/ASK-1, and the MAPKK SEK-1, along with the signaling scaffold protein TIR-1 [5,11-13]. The downstream kinase cascade behaves straightforwardly and cell-autonomously: a cell with high activity of the kinase homologs becomes AWCOFF and a cell with low gene activity becomes AWCON, regardless of the kinase activity in the contralateral AWC [11,12].

The results described above define a decision point between the nsy-5/nsy-4 multicellular signaling network and cell-autonomous execution of the decision by the kinase homologs. In previous studies, two genes encoding subunits of a CaV2-type voltage-activated calcium channel were shown to affect AWC asymmetry upstream of the kinases [5]. Loss-of-function mutants in unc-2, the CaV2 pore-forming α1 subunit, result in a mixed phenotype with wild-type, 2AWCON, and 2AWCOFF animals. By contrast, mutants in the regulatory α2δ subunit unc-36 have a pure, strong 2AWCON phenotype; the reason for this difference was unknown. Here we show that unc-2 cooperates with a second calcium channel α1 subunit, the CaV1 homolog egl-19, explaining the difference between unc-2 and unc-36. Rescue experiments and mosaic analysis provide evidence that the calcium channels act in cell communication between AWCs. The activity of the calcium channels is opposed by the AWC asymmetry gene olrn-1, which acts downstream of nsy-5 and nsy-4 in the induction of AWCON. olrn-1 represses the CaMKII/MAPK kinase cascade in AWCON and provides feedback to AWCOFF, coordinating the decisions of the left and right AWCs.

Results

The olrn-1 gene is expressed in AWC and promotes induction of AWCON

In a screen for AWC asymmetry mutants, we isolated the mutation olrn-1(ky626), which was named based on the isolation of another allele, olrn-1(ut305), from an olfactory learning screen [7]. olrn-1(ky626) animals had a highly penetrant 2AWCOFF phenotype (Figure 1a,b, Table 1), and they were able to chemotax to 2,3-pentanedione, an odor sensed by AWCOFF, but not butanone, an odor sensed by AWCON (Figure 1c). The AWC cell fate marker odr-1::dsRed was normally expressed in both AWCs of olrn-1 mutants, and AWC axon guidance was also apparently normal (Figure 1d). These results suggest that olrn-1(ky626) has one or more functional AWCOFF neurons and no AWCON neurons.

Table 1. str-2 expression in AWC in single and double mutants

thumbnailFigure 1. olrn-1 mutants have two AWCOFF neurons. (a,b) str-2::GFP expression in (a) wild type and (b) olrn-1(ky626) animals. Arrowhead, AWC; arrow, dim str-2::GFP expression in ASI. (c) Chemotaxis of wild-type and olrn-1(ky626) animals to the AWCOFF sensed odorant 2,3-pentanedione (pd) and the AWCON sensed odorant butanone (bu). A chemotaxis index of 1 indicates that 100% of animals approached the odorant, while a chemotaxis index of 0 represents random behavior. Error bars indicate standard error of the mean. (d) odr-1::DsRed is expressed in both AWC neurons (arrowheads) of olrn-1 mutants, a pattern identical to the wild-type pattern. (e,f) str-2::DsRed; srsx-3::GFP expression in (e) wild type and (f) olrn-1(ky626) mutant animals. Arrowheads indicate AWCs; dots indicate AWBs. Scale bars, 20 μm. Images are stacked confocal images.

A marker for the AWCOFF neuron provided further evidence that olrn-1 disrupts AWCOFF/AWCON asymmetry. srsx-3::GFP is expressed bilaterally in the AWB neurons [14] and asymmetrically in one of the two AWC neurons (Table 2). In wild-type animals expressing an srsx-3::GFP transgene and a str-2::dsRed transgene, the neuron expressing srsx-3::GFP was invariably contralateral to the neuron expressing str-2::dsRed (Figure 1e), indicating that srsx-3::GFP is expressed in AWCOFF. This interpretation was confirmed by mutant analysis: nsy-1(lf) (ASK1/MAPKKK) and unc-43(lf) (CaMKII) failed to express srsx-3::GFP in either AWC neuron, consistent with their 2AWCON phenotype, and unc-43(gf) expressed srsx-3::GFP in both AWC neurons, consistent with its 2AWCOFF phenotype (Table 2). olrn-1(ky626) mutants expressed srsx-3::GFP in both AWC neurons, suggesting that the neurons are both specified as AWCOFF (Figure 1f).

Table 2. srsx-3::GFP is expressed in AWCOFF

ky626 was mapped using single nucleotide polymorphisms to a small region on X and was determined to be an allele of olrn-1 by failure to complement olrn-1(ut305) (see Materials and methods). olrn-1(ut305) corresponds to the C02C6.2 gene [7]. C02C6.2 has two isoforms, C02C6.2a and C02C6.2b (olrn-1a and olrn-1b, respectively), which differ in their first 13 or 20 amino acids due to the use of alternative first exons (Figure 2a,b) [15]. A G to A mutation was identified in ky626 mutants at position 473 in the olrn-1a isoform (position 466 in the olrn-1b isoform), resulting in a missense mutation (G → E) in both isoforms (Figure 2b). olrn-1(ut305) is mutated at the splice acceptor site of the fourth intron [7] and, like olrn-1(ky626), results in a strong 2AWCOFF phenotype (99% penetrant, n = 91). A strain with a deletion of C02C6.2 has a lethal phenotype (data not shown); this lethality may be the null phenotype of olrn-1, or it may result from a linked mutation in another gene.

thumbnailFigure 2. olrn-1 encodes a protein with Raw repeats and potential transmembrane domains. (a) Genomic structure of olrn-1, showing alternative first exons for a and b isoforms, whose 5' ends are separated by 3.8 kb. (b) Translation of olrn-1, showing alternative first exons for olrn-1a and olrn-1b isoforms. Two short repeats shared with Drosophila Raw are highlighted in green with conserved residues in bold type; potential transmembrane domains are boxed. The location of the splice acceptor site (fourth exon) mutated in ut305 and the residue mutated in olrn-1(ky626) (G 473a/466b E) are marked. Arrow marks the carboxy-terminal insertion site of Cherry in odr-3::olrn-1b::Ch (f). (c) Expression of olrn-1a::Cherry promoter fusion. Arrowhead indicates Cherry expression in AWC neuron expressing str-2::GFP. (d) Expression of olrn-1b::Cherry promoter fusion in non-neuronal cells. Arrows, hypodermal cells. The pharynx is a prominent site of expression. Arrowhead, no Cherry expression in AWC neuron expressing str-2::GFP. (e) Expression of amino-terminally tagged odr-3::Cherry::olrn-1b in an L4 olrn-1(ky626) animal. odr-3::Cherry::olrn-1b is excluded from the nucleus and is punctate in the axon and dendrite. (f) Expression of carboxy-terminally tagged odr-3::olrn-1b::Cherry in an L4 ky626 animal. odr-3::olrn-1b::Cherry is excluded from the nucleus, and is punctate in the axon and dendrite. Arrowheads, AWC cell bodies. Scale bars, 20 μm. Images are stacked confocal images.

Expression of olrn-1 cDNAs under the AWC-selective odr-3 promoter rescued the 2AWCOFF phenotypes of olrn-1(ky626) mutants [7] (Table 3). Additionally, overexpression of olrn-1b in a wild-type background caused a 2AWCON phenotype (olrn-1a was not tested; Tables 1a and 3). These results indicate that high olrn-1 activity promotes the AWCON phenotype, support AWC as the likely site of olrn-1 function, and suggest that ky626 and ut305 are reduction-of-function alleles.

Table 3. Rescue of olrn-1, overexpression of olrn-1, and effect of mutations

To establish the potential olrn-1 expression pattern, two regions upstream of olrn-1 were fused to coding sequences for the fluorescent protein mCherry [16]. The 3.8 kb region upstream of the olrn-1a start site was expressed in AWC neurons as well as ASG and BAG sensory neurons (Figure 2c). The 3.6 kb region upstream of the olrn-1b start site was expressed in the marginal cells of the pharynx, anterior hypodermal cells and the rectal gland cells (Figure 2d and data not shown). Although individual promoter fragments may not reproduce the entire olrn-1 expression pattern, these results suggest that olrn-1 may normally be expressed in AWC and in other cells.

The first Raw repeat and a carboxy-terminal region are important for OLRN-1 function

OLRN-1 is a previously uncharacterized protein that is conserved along its entire length with related proteins from Caenorhabditis remanei and Caenorhabditis briggsae. It bears more distant similarity with the Drosophila melanogaster gene raw (or cyrano). raw restricts JNK signaling during dorsal closure of the fly embryo, and raw mutants have an embryonic dorsal-open phenotype resulting from abnormal cell migration, as well as nervous system defects [17]. The similarity between ORLN-1 and Raw is highest in two repeated domains of unknown function [17] (Figure 2b). OLRN-1 has a bipartite, highly hydrophobic region of approximately 40 amino acids at residues 264–280 and 288–304 that is likely to mediate membrane attachment; this domain is not present in Raw. One possibility is that these two hydrophobic domains form a hairpin-like transmembrane domain, so that both the amino and carboxyl termini of OLRN-1 face the cytoplasm.

The predicted OLRN-1B protein was tagged at its amino or carboxyl terminus by inserting mCherry into the odr-3::olrn-1 vector. Both amino- and carboxy-terminally tagged OLRN-1 rescued olrn-1(ky626) mutants (Table 3 and data not shown). The tagged OLRN-1b proteins were localized to punctate structures in the axon, dendrite, and cell body, but largely excluded from nuclei (Figure 2e,f).

Structure-function analysis of OLRN-1 was conducted to identify important domains of the protein (Figure 3a). Deletions in the odr-3::olrn-1b::Cherry rescuing clone were made to remove the predicted Raw repeats (ΔrawR1, ΔrawR2), the transmembrane domains (ΔTM1,2) and the region carboxy-terminal to the second Raw repeat (ΔCterm). A set of four adjacent arginines reminiscent of a cleavage or nuclear localization signal was deleted from the carboxyl terminus (ΔRRRR). Finally, the G466E ky626 mutation was engineered into the full-length protein to examine the properties of the mutated protein. All of these Cherry-tagged mutant DNAs were cloned under the AWC-selective odr-3 promoter and introduced into olrn-1(ky626) mutants at 15 ng/μl, a concentration that resulted in a 2AWCON phenotype in approximately 80% of animals carrying a wild-type odr-3::olrn-1b::mCherry transgene (Figure 3b, Table 3). Deletion of the first Raw repeat (ΔrawR1) nearly eliminated the activity of olrn-1, as did deletion of the carboxy-terminal region (ΔCterm). By contrast, deletions of the transmembrane domains (ΔTM1,2) or the second Raw repeat (ΔrawR2) did not greatly diminish the activity of olrn-1 (Figure 3b, Table 3). Transgenes carrying the deletion of the four arginines (ΔRRRR) and the G466E missense mutation were intermediate in activity. ΔRRRR and G466E transgenes were able to rescue olrn-1(ky626), but did not cause the overexpression phenotype caused by the full-length olrn-1 transgene (Figure 3b). Their activity was similar to that of full-length odr-3::olrn-1 injected at six-fold lower DNA concentrations (Table 3). These results suggest that ΔRRRR and G466E mutations reduce the activity of the OLRN-1 protein.

thumbnailFigure 3. Structure-function analysis of odr-3::olrn-1::Cherry. (a) Mutants generated in odr-3::olrn-1b::Cherry affected Raw repeats (ΔrawR1, ΔrawR2), potential transmembrane domains (ΔTM1,2), the four adjacent arginines (ΔRRRR), and a carboxy-terminal region (ΔCterm). The ky626 mutation (G466E) was also introduced. (b) Phenotypes of ky626 animals expressing odr-3:olrn-1b::Cherry transgenes. olrn-1(ky626) control is at left. All transgenes except ΔCterm showed significant rescue compared to nontransgenic sibling controls; all transgenes except rawR2 were significantly less active than intact odr-3::olrn-1b::Cherry (P < 0.008 by Fisher exact test or Chi square test as appropriate; 0.008 was used as the significance level based on the conservative Bonferroni correction for six comparisons; n > 100 rescued animals per clone, from at least two independent transgenic lines that showed similar degrees of rescue (Table 3)).

The expression levels and localization of OLRN-1b were examined in olrn-1(ky626) animals bearing the mutated clones. All mutated clones produced comparable levels of OLRN-1::Cherry fluorescence in AWC, suggesting that the defective mutants made dysfunctional but stable proteins (Additional file 1). It was not possible to resolve the subcellular localization of OLRN-1 in embryos, but immediately after hatching the OLRN-1b::Cherry protein was present in the AWC cell body, axon and dendrite (Additional file 1). All mutant proteins had similar localization to wild-type OLRN-1b::Cherry (Additional file 1). Twelve hours later during the late L1/early L2 stage, wild-type OLRN-1b::Cherry and most mutant proteins were still localized to the AWC cell body, axon, and dendrite, but OLRN-1b(ΔTM1,2) was no longer detectable in axons (Additional file 1).

Additional File 1. Subcellular localization of wild-type and mutant olrn-1b::Cherry proteins. Confocal images of odr-3::olrn-1b::Cherry proteins in olrn-1(ky626) animals at (a-g) 0–3 hours after hatching and (h-n) L1/L2 stage, 20 hours after hatching. Notched arrowheads indicate AWC axons, flat arrowheads indicate AWC cell bodies. The diagrams show the approximate size and disposition of AWC neurons in the images (anterior is at left). (a-g) Two AWCs are visible in most images; (h-n) only one AWC is visible in most images, but both AWCs have similar OLRN-1b:Cherry levels. Scale bars are 10 μm.

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olrn-1 antagonizes calcium pathways in AWC signaling

The olrn-1(ky626) mutation and the olrn-1(OE) overexpressing transgene were combined with other mutations to ask how olrn-1 interacts with AWC asymmetry genes. We first examined the upstream signaling genes, the claudin-like nsy-4 and the innexin nsy-5 [9,10]. Loss-of-function mutations in nsy-4, nsy-5 and olrn-1 that caused 2AWCOFF phenotypes were combined with overexpressing transgenes for nsy-4, nsy-5 and olrn-1 that caused 2AWCON phenotypes. In all combinations, the double mutants resembled the olrn-1 parent more closely than the nsy-4 or nsy-5 parent (Table 1a), but mixed phenotypes were observed. These results suggest that olrn-1 acts mainly at a step downstream of nsy-4 and nsy-5, but the absence of definitive null alleles of nsy-4 and olrn-1 limits this interpretation.

Loss-of-function mutations in the unc-2 α 1 or unc-36 α 2δ calcium channel subunits result in a strong (unc-36) or mixed (unc-2) 2AWCON phenotype. Both unc-36 olrn-1 double mutants and unc-2 olrn-1 double mutants resembled olrn-1 single mutants, with a high fraction of 2AWCOFF animals (Table 1a). Different results were observed in double mutants between olrn-1 and loss-of-function mutations in the CaMKII/MAP kinase cascade – the kinase genes unc-43 (CaMKII), nsy-1 (MAPKKK), and sek-1 (MAPKK) [5,11,12]. Double mutants between olrn-1 and the three kinases invariably resembled the kinase mutants, with a strong 2AWCON phenotype (Table 1a). tir-1 olrn-1 double mutants had a mixed, nearly wild-type phenotype (Table 1a), but as neither gene has definitive null alleles, the significance of these results is unclear. These results suggest that olrn-1 acts between the calcium channels and the CaMKII/MAP kinase cassette (see Discussion).

unc-2 CaV2 and egl-19 CaV1 calcium channel homologs act together in AWC asymmetry

The suggestion that olrn-1 acts at a genetic step near the voltage-activated calcium channel homolog unc-2 prompted a more detailed examination of unc-2 and unc-36. Previous studies showed that the putative CaV2 null mutant unc-2(e55) had a mixed AWC phenotype with 30–60% 2AWCON animals and 4–25% 2AWCOFF animals (Table 1a) [5]. unc-2(lj1), a second strong loss-of-function mutant, shared this mixed phenotype (Table 1b), but null mutants for the channel-associated α2δ subunit unc-36 had a strong 2AWCON phenotype [5] (Table 1b; see Materials and methods for molecular analysis of unc-36(e251) and unc-2(e55)). These results could be explained if multiple α1 subunits participate in AWC asymmetry, sharing the unc-36 α2δ subunit. The C. elegans genome encodes five proteins related to α1 subunits, including one CaV1 subunit and one CaV2 subunit. Null mutations in the CaV1 homolog egl-19 are embryonic lethal, and partial loss-of-function mutations have normal AWC asymmetry [18] (Table 1b). When egl-19 partial loss-of-function alleles were combined with null alleles of unc-2, the double mutants had a strong 2AWCONphenotype reminiscent of unc-36 mutants (Table 1b). These results are consistent with partially redundant functions between egl-19 and unc-2, with both channels contributing to AWC asymmetry.

Calcium channels can serve as scaffolding proteins in addition to their ion-conducting properties. The ion-conducting properties of EGL-19 are affected by the gain-of-function mutation egl-19(ad695gf), which decreases channel desensitization [18,19]. egl-19(ad695gf) unc-2(lf) mutants had a strong 2AWCOFFphenotype, the opposite phenotype from the egl-19(lf) unc-2(lf) mutants (Table 1b). This result suggests that the ion-conducting activity of EGL-19 contributes to its activity.

The strong 2AWCOFF phenotype of egl-19(ad695gf) unc-2(lf) was not observed in egl-19(ad695gf) single mutants (Table 1b). Thus, egl-19(gf) has an activity in AWC that is masked by the normal activity of the unc-2 gene, but revealed when unc-2 is eliminated (see Discussion).

olrn-1 acts in the future AWCON neuron, and unc-2/unc-36 act in the future AWCOFF neuron, to coordinate AWC signaling

Genetic mosaic analysis is a useful approach for distinguishing between the two AWC neurons as they signal and respond to each other in development. Any gene in the asymmetry pathway could, in principle, function in the future AWCOFF cell or in the future AWCON cell. Two kinds of results have been observed in previous genetic mosaic studies. The nsy-4 claudin and nsy-5 innexin genes act cell-autonomously to promote induction of AWCON, and also act cell non-autonomously to prevent AWCON induction in the contralateral AWC [9,10]. At this signaling stage, each AWC appears to monitor the activity state of the other. By contrast, the kinases unc-43 and nsy-1, and the scaffold tir-1 act strictly cell-autonomously: a cell with high kinase activity becomes AWCOFF, and a cell with low kinase activity becomes AWCON[11,12]. At this execution stage, the decision has been made and the AWCs are independent. To understand how the decision is made, we used genetic mosaic analysis to examine animals in which the two AWCs had different levels of olrn-1,unc-2 and unc-36 gene activity (Figures 4 and 5). Unstable extrachromosomal arrays containing the AWC marker odr-1::DsRed and either odr-3::olrn-1, odr-3::unc-2, or odr-3::unc-36 test plasmids were introduced into strains with stable expression of the AWCON marker str-2::GFP (Figures 4a,c and 5b,d,f,h). Random loss of the arrays from one AWC was detected using the odr-1::DsRed marker, and then both AWC neurons were scored for the expression of the str-2::GFP AWCON marker. The methods for these experiments were similar to those used in previous studies; control experiments indicate that the promoters and markers do not affect AWC asymmetry (see Materials and methods) [10-12].

thumbnailFigure 4. olrn-1 mosaic analysis. (a) Rescue of olrn-1(ky626) by odr-3::olrn-1b injected at 2.5 ng/μl, 5 ng/μl, or 15 ng/μl with odr-1::DsRed. (b) AWC phenotypes of mosaic olrn-1(ky626) animals that express odr-3::olrn-1b odr-1::dsRed transgene in one AWC. (c) Phenotypes in wild-type animals overexpressing odr-3::olrn-1b injected at 15 ng/μl or 25 ng/μl with odr-1::DsRed. (d) AWC phenotypes of wild-type mosaic animals that overexpress odr-3::olrn-1b in one AWC. For statistical analysis, see Materials and methods. (e) Phenotypes of nsy-4 olrn-1(OE) and nsy-5 olrn-1(OE) strains and controls. (f) AWC phenotypes of mosaic animals that express odr-3::olrn-1b in one AWC in nsy-4 or nsy-5 mutant backgrounds.

thumbnailFigure 5. Mosaic analysis of the unc-36/unc-2 calcium channel genes. (a) Rescue of unc-2(lj1) by [odr-3::unc-2, odr-1::dsRed] array. Green, str-2::GFP expression; red, odr-1::dsRED expression. Arrows point to AWCON neurons. (b) Rescue of unc-36(e251) phenotypes in three [odr-3::unc-36, odr-1::dsRed] transgenic lines. (c) AWC phenotypes of unc-36 mosaic animals that express odr-3::unc-36 in one AWC. (d) The three [odr-3::unc-36, odr-1::dsRed] transgenes from (b) were introduced into a wild-type background. (e) AWC phenotypes of wild-type mosaic animals that overexpress odr-3::unc-36 in one AWC. (f) Rescue of unc-2(lj1) phenotypes in three Ex [odr-3::unc-2, odr-1::dsRed] transgenic lines. (g) AWC phenotypes of unc-2 mosaic animals that express odr-3::unc-2 in one AWC. (h) The three [odr-3::unc-2, odr-1::dsRed] transgenes from (f) were introduced into a wild-type background. (i) AWC phenotypes of wild-type mosaic animals that overexpress odr-3::unc-2 in one AWC. n, number of animals scored. For statistical analysis, see Materials and methods.

In an olrn-1 mutant background, most mosaic animals with a single rescued AWC had the wild-type, asymmetric phenotype: the rescued cell became AWCON and the mutant cell became AWCOFF (Figure 4b). This result suggests that olrn-1 acts cell autonomously in the future AWCON cell to induce its identity. In wild-type animals overexpressing odr-3::olrn-1 transgenes, most animals that lost the transgene in one AWC also had a wild-type asymmetric AWC phenotype. In these animals, the cell overexpressing olrn-1 nearly always became AWCON, and the wild-type contralateral cell nearly always became AWCOFF (Figure 4d). This behavior is unlike the behavior of fully wild-type animals in which each cell becomes AWCON or AWCOFF at equal frequencies. The wild-type mosaics suggest that a cell with higher olrn-1 expression can prevent the contralateral cell from becoming AWCON, and therefore implicate olrn-1 in feedback between AWCs. Very similar results were previously obtained in mosaic analysis of nsy-4 and nsy-5 [9,10].

The results described above might be confounded by nsy-4- and nsy-5-dependent signaling between AWCs. To separate cell-intrinsic functions of olrn-1 from possible network functions, we generated mosaics overexpressing OLRN-1 in one AWC in nsy-4 and nsy-5 mutants (Figure 4e,f). In these experiments,olrn-1(OE) behaved exactly as it did in the wild-type background, specifically converting the olrn-1-expressing neuron to AWCON (Figure 4f). These results suggest that olrn-1 functions independently of, and most likely downstream of,nsy-4 and nsy-5 in AWCON.

Similar rescue and mosaic overexpression experiments were conducted with the calcium channel genes unc-36 (α2δ subunit) and unc-2 (CaV2 α1 subunit). Expression of either gene under the AWC-selective odr-3 promoter rescued AWC asymmetry of the corresponding mutant (see Materials and methods; Figure 5a,b,f). Thus, the calcium channels are likely to function within AWC neurons. A mild gain-of-function phenotype was observed with odr-3::unc-2, but not with odr-3::unc-36 (Figure 5d,h).

For both unc-36 and unc-2, mosaic animals with a single rescued AWC had the wild-type asymmetric phenotype: the rescued cell always became AWCOFF, and the mutant cell always became AWCON (Figure 5c,g). This result was substantially different from the result with downstream kinases such as unc-43, where a single rescued AWC randomly became AWCON or AWCOFF, and the mutant cell always became AWCON [12]. The difference suggests that unc-36 and unc-2 influence the coordinated AWCON/AWCOFF decision, whereas unc-43 and other kinases act to execute a decision that has already been made. The rescue of the unc-2 mutant cell in these mosaics is particularly informative. unc-2 has an incompletely penetrant phenotype; in unc-2 controls, 33% of animals have one AWCOFF neuron, and 4% have two AWCOFF neurons, so in total approximately 20% of all unc-2 mutant AWCs became AWCOFF (Table 1b). In unc-2 mosaics with one rescued AWC, fewer than 5% of the unc-2(-) AWCs became AWCOFF, indicating that the unc-2(-) AWC neuron was affected by the rescued AWC on the contralateral side.

Only a mild overexpression phenotype was observed upon introduction of odr-3::unc-2 into wild-type animals (Figure 5h), suggesting that the tightly regulated calcium channels may be relatively resistant to variations in expression levels. In mosaic animals in which only one AWC overexpressed odr-3::unc-36 or odr-3::unc-2 in a wild-type background, the overexpressing AWCs or the contralateral AWCs were equally likely to become AWCON or AWCOFF (Figure 5e,i). These results indicate that unlike nsy-4,nsy-5, and olrn-1, relative unc-2 and unc-36 expression levels are not critical to the AWCON/AWCOFF decision.

Discussion

This analysis adds two genes to the pathway for AWC asymmetry: the gene olrn-1, and the C. elegans CaV1 homolog egl-19, whose cooperation with unc-2 explains the weak phenotype of the unc-2 (CaV2) mutant. These genes and other genes in the AWC asymmetry pathway have been classified in three ways: double mutant analysis, which can reveal biological regulatory relationships; targeted rescue and mosaic analysis to determine the essential cellular site of expression; and detailed mosaic analysis to determine whether expression of the gene in one AWC affects the contralateral AWC. Combining these three approaches suggests that the coordinated decision between AWCON and AWCOFF occurs at the interface between the calcium channels (UNC-2/UNC-36/EGL-19) and OLRN-1.

In C. elegans AWC neurons, olrn-1 has genetically defined functions that are similar to those of the innexin gene nsy-5 and the claudin/calcium channel γ subunit gene nsy-4. All three genes are required for the induction of AWCON, and all have similar cell-autonomous and non-autonomous effects on AWC in mosaic analysis [9,10].olrn-1 overexpression induced AWCON cell-autonomously in nsy-4 and nsy-5 mutants, suggesting that olrn-1 acts downstream of these two genes or independently of them in AWCON. The nature of any olrn-1 regulation by the upstream genes is unknown. There were no obvious effects of olrn-1 mutations on tagged NSY-4 or NSY-5 proteins in AWC, nor were there obvious effects of nsy-4 or nsy-5 on tagged OLRN-1 protein (data not shown).

olrn-1 mutations were epistatic to null mutations in the calcium channel genes unc-2 and unc-36, whereas calcium channel null mutations are epistatic to nsy-4 and nsy-5 [9,10]. The behavior of olrn-1 in these double mutants supports the suggestion that it acts at a later step in signaling than nsy-4 and nsy-5. Epistasis analysis does not provide detailed molecular mechanisms, and some conclusions are not firm when null alleles are unavailable. However, since the unc-2 and unc-36 mutations are molecular nulls, the epistasis result proves that these channel genes are not essential for olrn-1 activity.

The kinase mutations unc-43 (CaMKII), nsy-1 (ASK1/MAPKKK), and sek-1 (MAPKK) were fully epistatic to olrn-1 mutations. Thus, at a genetic level, olrn-1 may prevent unc-2 from activating the CamKII homolog unc-43 in the future AWCON. In molecular terms, this might mean that OLRN-1 inhibits calmodulin (which would act at this step) or that OLRN-1 prevents calcium from UNC-2 channels from activating UNC-43, perhaps by binding the channel or the kinase (Figure 6). There is no evidence for direct interactions between these proteins, and many other possibilities exist. olrn-1 is related to Drosophila raw/cyrano, which is required for epithelial movements that drive dorsal closure of the fly embryo, for epithelial morphogenesis, and for neuronal development [17,20]. An olrn-1 domain that is similar to raw is needed for full olrn-1 activity, supporting the significance of the homology. The direct targets of Raw are unknown, but raw mutants have excessive phospho-Jnk during dorsal closure, suggesting that Raw inhibits Jnk MAP kinase pathways [17]. This biochemical evidence that Raw is a kinase inhibitor parallels our genetic conclusion that olrn-1 directly or indirectly inhibits the kinase pathway consisting of unc-43 (CaMKII), nsy-1 (ASK1, a p38/Jnk MAPKKK), and sek-1 (MAPKK).

thumbnailFigure 6. Model for calcium channel function and OLRN-1 in the AWCON/AWCOFFdecision. All genes are expressed both in the left and in the right AWCs; color is used to indicate the cell in which each gene product is more active. The future AWCOFF transmits a signal to AWCON via NSY-5 gap junctions between AWC and other cells and NSY-4 claudins. This signal might be membrane potential. In AWCON, the signal suppresses the UNC-2 (CaV2) and EGL-19 (CaV1) voltage-activated calcium channels and allows high OLRN-1 activity. OLRN-1 inhibits the UNC-43 (CaMKII)/NSY-1/SEK-1 kinase cascade cell-autonomously within AWCON. A feedback signal from the calcium channels and OLRN-1 is transmitted from AWCON back to AWCOFF.

The analysis of egl-19 CaV1 mutations underscores the importance of calcium channels in AWC asymmetry. unc-2 CaV2 mutants have a weak and mixed phenotype, raising initial doubts about the significance of the channel, but the highly penetrant 2AWCON phenotype of egl-19 unc-2 double mutants suggests that calcium entry through voltage-activated calcium channels is essential for specification of AWCOFF. The strong, opposite 2AWCOFF phenotype of the egl-19(gf) unc-2(lf) double mutant further suggests that sufficient calcium entry through EGL-19 can act instructively to specify AWCOFF. This phenotype was not observed in egl-19(gf) single mutants, suggesting that unc-2 inhibits the activity of egl-19(gf). CaV channels generate calcium and voltage signals, and are subject to calcium- and voltage-dependent activation and inactivation, so there are many levels at which channel cross-regulation could take place [21].

The phenotype of unc-36 in AWC asymmetry suggests that this CaV α2δ subunit promotes the activity of both unc-2 and egl-19 α1 subunits. Previous calcium imaging studies of C. elegans pharyngeal muscles suggested that unc-36 inhibits egl-19 [19], whereas calcium imaging in mechanosensory neurons suggested that unc-36 activates egl-19 [22]. We suggest that both previous observations are correct, and that in pharyngeal muscles, as in AWC, unc-2/unc-36 channels inhibit egl-19 or egl-19/unc-36 channels.

A model for the functions of olrn-1, unc-2, and unc-36 in the signaling pathway, based on this work and prior work, is presented in Figure 6. Induction of AWCON from an AWCOFF-like ground state requires cooperation between the innexin gene nsy-5, which assembles a multicellular gap junction network and preferentially induces AWCR to the AWCON state, and the claudin/γ-subunit like nsy-4, which preferentially induces AWCL to the AWCON state [9,10]. Tight junctions (which contain claudins) and gap junctions (which are composed of innexins) potentiate one anothers' activity in epithelia, providing a possible analogy for the nsy-4/nsy-5 cooperation in AWC [23]. A signal must be transmitted by this multicellular network; the strong involvement of the voltage-regulated calcium channel homologs unc-2,egl-19, and unc-36 in AWC asymmetry suggests that the signal regulates membrane potential. Voltage changes are efficiently transmitted through gap junctions, whereas calcium is poorly diffusible and is, therefore, transmitted inefficiently. Thus, voltage signals from UNC-2, EGL-19, and possibly other channels could be transmitted from AWCOFF to AWCON via gap junctions. At least two other voltage-regulated channels, the potassium channels SLO-1 and EGL-2, also affect AWC asymmetry [5,24]. The appeal of this model is that voltage-regulated channels such as unc-2/unc-36 could act both to generate a signal in one AWC and to detect the signal in the contralateral AWC.

The coordinated decision to form one AWCON and one AWCOFF requires a symmetry-breaking event. Like many genes in the AWC asymmetry pathway, unc-2/unc-36 activity is predicted to be high in one AWC, and low in the other; unlike other genes, there are plausible mechanisms by which a symmetry-breaking event could differentially regulate calcium channels. An interesting example is provided by the pacemaker cells of the vertebrate heart, which are found in the sinoatrial (SA) node. Gap junctions and voltage-activated calcium channels are essential to the synchronization of SA pacemaker cells and the generation of a coherent heartbeat [25]. Isolated SA cells have rhythmic action potentials that are driven by calcium channels and other conductances. When two SA cells come into contact, they form gap junctions that lead to synchronization of the two cells, at a rhythm that is dominated by the faster, or leader, cell. Two synchronized SA cells would appear to be similar, but in fact, the result of their synchronization is a coupling of membrane potential and an uncoupling of individual conductances within the two cells [26]. During the diastolic period between heartbeats, the leader cell has an ongoing inward current, while the follower cell has an outward current [26]. In other words, the leader cell experiences inward currents both at the beginning of the calcium action potential and in the long period between action potentials; the follower cell experiences inward currents only during the action potential. As calcium-activated signaling pathways are exquisitely sensitive to the temporal pattern of calcium signals [27,28], different patterns of inward calcium currents in two cells have the potential to create sustained differences between them.

We suggest that in isolation, both AWCs have spontaneous activity sufficient to maintain CaMKII activity and the AWCOFF state. When gap junctions form via NSY-5, the spontaneous activity of the AWCs is coupled, and by analogy to the SA node, one cell leads and the other follows. The leader cell maintains ongoing calcium entry and becomes AWCOFF; gap junction coupling reduces calcium entry into the follower cell, and it becomes AWCON. In this model, the calcium channels have both an effector function (calcium entry and activation of CaMKII) and a signaling function (altering membrane potential). We speculate that similar mechanisms may operate in many developing nervous systems during the transient period that gap junctions are prominent. Since it acts at a similar step, olrn-1 could affect either the propagation of the signal or its effectiveness in the responding cell.

Gap junctions, claudins, and membrane potential affect left-right asymmetry of the Xenopus body axis, suggesting a possible molecular similarity between vertebrate asymmetry and the pathways that regulate C. elegans AWC neurons [29-31]. Although the later nodal/Shh pathways used in vertebrate left-right patterning are different from those used in AWCs, there may be hidden similarities that remain to be discovered. The left-right asymmetry of the human brain is more variable than the human body plan; left-handedness and reversed lateralization of language areas are much more common than inversion of the internal organs [32]. Lateralized neurological disorders such as migraine headaches and Rasmussen encephalitis randomly affect one side of the brain, providing indirect evidence of variably asymmetric brain functions [33,34]. An asymmetric migraine syndrome in humans is caused by mutations in CaV2 calcium channels, which are orthologs of unc-2 [33]. Further analysis of asymmetric brain function in humans may reveal unexpected connections with the asymmetric nervous system of C. elegans.

Materials and methods

Strains

Wild-type strains were C. elegans variety Bristol, strain N2. The CB4856 strain was used for mapping olrn-1 [35]. Strains were maintained by standard methods [36].

Germline transformation was carried out as previously described [37]. Co-injection markers were ofm-1::GFP,ofm-1::RFP or elt-2::GFP. Integrated transgenes used in this study included kyIs140 I [str-2::GFP, lin-15(+)], kyIs323 II [str-2::GFP, ofm-1::GFP], zdIs5 [mec-4::GFP]. Mutations used in this study included LG (linkage group) I: nsy-5(ky634), LGII: nsy-1(ky542), nsy-1(ag3),nsy-1(ok593), LGIII: tir-1(tm1111), unc-36(e251), LGIV: egl-19 (n582), egl-19(ad695gf), nsy-4(ky616), unc-43(n1186), unc-43(n408), unc-43(n498gf), LGX: olrn-1(ky626),olrn-1(ut305), unc-2 (lj1),unc-2(e55), sek-1(km4).

Transgenes maintained as extrachromosomal arrays included the following lines used for mosaic analysis: kyEx914 (line 1) & kyEx918 (line 2) [odr-3::olrn-1b 15 ng/ul, odr-1::dsRed 7.5 ng/ul, ofm-1::gfp 20 ng/ul], kyEx1072 (line 1) & kyEx1074 (line 2) [odr-3::olrn-1b 5 ng/ul, odr-1::dsRed 7.5 ng/ul, ofm-1::gfp 20 ng/ul], kyEx1097 (line 1) & kyEx1098 (line 2) [odr-3::olrn-1b 2.5 ng/ul, odr-1::dsRed 7.5 ng/ul, elt-2::GFP 10 ng/ul], kyEx1102 (line 1) & kyEx1103 (line 2) [odr-3::olrn-1b 25 ng/ul, odr-1::dsRed 7.5 ng/ul, elt-2::GFP 5 ng/ul]. For unc-2 and unc-36 mosaics, the same extrachromosomal arrays were examined in wild-type and mutant backgrounds: kyEx1628 (line 1) kyEx1629 (line 2) and kyEx1630 (line 3) [odr-3::unc-2 20 ng/ul, odr-1::RFP 2.5 ng/ul, ofm-1::GFP 10 ng/ul]; kyEx1229 (line 1) kyEx1387 (line 2) and kyEx1388 (line 3) [odr-3::unc-36 20 ng/ul, odr-1::RFP 2.5 ng/ul, elt-2::GFP 10 ng/ul].

Additional transgenic arrays were kyEx822 [odr-3::nsy-4a 75 ng/ul, ofm-1::GFP 20 ng/ul], kyEx996 [18.5 kb nsy-5 PCR fragment 13 ng/ul, odr-1::DsRed 12.5 ng/ul, ofm-1::GFP 25 ng/ul], kyEx1075 [srsx-3::GFP 10 ng/ul, str-2::DsRed 50 ng/ul, elt-2::gfp 10 ng/ul], kyEx1096 [odr-1::dsRed 7.5 ng/ul, elt-2::GFP 10 ng/ul], kyEx1182 [odr-3::olrn-1b::Ch 5 ng/ul, ofm-1::GFP 15 ng/ul], kyEx1320 [olrn-1a::Ch 25 ng/ul, ofm-1::GFP 15 ng/ul], kyEx1315 [olrn-1b::Ch 50 ng/ul, elt-2::GFP 10 ng/ul], kyEx1310 [odr-3::Ch::olrn-1b 25 ng/ul, elt-2::GFP 10 ng/ul].

The following arrays were used in the deletion analysis: odr-3::olrn-1b::Ch = kyEx1318 (line 1), kyEx1319 (line 2), kyEx1317 (line 3); odr-3::olrn-1bΔ rawR1::Ch = kyEx1337 (line 1), kyEx1345 (line 2), kyEx1344 (line 3); odr-3::olrn-1bΔ TM1,2::Ch = kyEx1297 (line 1), kyEx1298 (line 2); odr-3::olrn-1bΔ rawR2::Ch = kyEx1338 (line 1), kyEx1339 (line 2), kyEx1340 (line 3); odr-3::olrn-1b(G466E)::Ch = kyEx1358 (line 1), kyEx1357 (line 2), kyEx1359 (line 3); odr-3::olrn-1b(ΔRRRR)::Ch = kyEx1332 (line 1), kyEx1296 (line 2), kyEx1300 (line 3), kyEx1299 (line 4); odr-3::olrn-1bΔ Cterm::Ch = kyEx1382 (line 1), kyEx1352 (line 2), kyEx1351 (line 3). These transgenes were injected at 15 ng/μl with 10–15 ng/μl of ofm-1::GFP co-injection marker.

Isolation and characterization of olrn-1(ky626)

kyIs140 I animals were subjected to ethyl methane sulfonate (EMS) mutagenesis according to standard procedures [38]. A chemotaxis enrichment was used to isolate F2 animals that sensed 2,3 pentanedione (an AWCOFF-sensed odorant) but failed to sense butanone (an AWCON-sensed odorant) [10]. F2 mutants that failed to migrate to butanone were screened for the AWCOFF str-2::GFP phenotype using a fluorescence dissecting microscope. The failure to express str-2::GFP was confirmed using 400× magnification under a compound microscope. Additional rounds of screening were done without the behavioral enrichment.

olrn-1(ky626) was mapped on LGX between single nucleotide polymorphisms pkP6166 (physical position X: 14678988) and pkP6172 (physical position X:17707311) using the CB4856 strain [35]. A complementation test between ky626 and ut305 resulted in a failure to complement: 93.5% of ky626/ut305 (n = 138) animals at 25°C were 2AWCOFF. olrn-1(ut305) has previously been shown to correspond to C02C6.2 (X:15539330–15546729) [7]. To identify the olrn-1(ky626) mutation, genomic coding regions of C02C6.2 were amplified by PCR and sequenced on both strands. The olrn-1(ky626) mutation was a G → A transition, resulting in a G → E missense mutation at residue 473 in the olrn-1a isoform, and position 466 in the olrn-1b isoform.

Molecular biology

Identification of unc-2(e55) and unc-36(e251) mutations

Resequencing unc-2(e55) revealed that the stop mutation originally assigned to residue 458 was actually present at reside 511 of the T02C5.5b gene model (Q>stop nonsense mutation), but supported the identification of e55 as a strong loss-of-function mutation.

Sequencing of unc-36(e251) revealed that the unc-36(e251) mutation was a G → A transition, resulting in a Trp>stop nonsense mutation at residue 496 in the C50C3.9a gene model. This mutation should truncate the UNC-36 protein immediately after the vWA domain.

odr-3::unc-2

unc-2 cDNAs were obtained by PCR from a C. elegans cDNA library using primers flanking the open reading frame T02C5.5b. Due to toxicity of the full-length cDNAs in bacteria, they were maintained as minigenes with a synthetic intron interrupting their coding regions. When expressed from the pan-neuronal H20 promoter, the unc-2 minigene rescued the uncoordinated phenotype of unc-2(lj1). The minigene was subcloned behind the odr-3 promoter for mosaic analysis.

odr-3::unc-36

unc-36 cDNAs were obtained by PCR from a C. elegans cDNA library using primers flanking the open reading frame C50C3.9a. Expression of the cDNA under 2 kb of unc-36 upstream region rescued the uncoordinated phenotype of unc-36(e251). The cDNA was subcloned behind the odr-3 promoter for mosaic analysis.

odr-3::olrn-1b

odr-3::olrn-1b was constructed by inserting a KpnI-C02C6.2b-SmaI fragment into the pPD49.26 vector at the KpnI and EcoRV sites. A KpnI-C02C6.2b-ApaI fragment from this clone was then inserted downstream of the odr-3 promoter in the odr-3::GFP vector [39] removing the green fluorescent protein (GFP).

odr-3::mCherry::orln-1b & odr-3::olrn-1b::mCherry

The mCherry coding sequence was amplified using primers with linkers on each side of mCherry, and subcloned into either the 5' NheI site upstream of the olrn-1b start site or an internal EcoRV site in the carboxyl terminus in the odr-3::olrn-1b vector. This insertion site was carboxy-terminal to the second Raw repeat domain (rawR2).

odr-3::olrn-1b deletions

To make the domain deletions described in Figure 3, the odr-3::olrn-1b::Cherry vector was deleted at residues 77–108 (ΔrawR1), 265–304 (ΔTM1,2), 396–428 (ΔrawR2), 510–513 (ΔRRRR), and 429–539 (ΔC-term) by PCR overlap extension with suitable primers [40,41]. The same technique was used to introduce the G466E mutation.

olrn-1a::mCherry and olrn-1b::mCherry

3.8 kb 5' to the olrn-1a start site and 3.6 kb 5' to the olrn-1b start site were subcloned into the pSM-mCherry vector. The second clone represented the entire intron between the alternative first exons of the OLRN-1 isoforms.

Genetic mosaic analysis

Loss of function mosaic analysis was performed on six independent lines with unstable extrachromosomal transgenic arrays [odr-3:olrn-1b, odr-1::dsRed] in a kyIs140 I, olrn-1(ky626) X mutant. Gain-of-function mosaic analysis was performed on four independent lines in a kyIs140 I wild-type strain. Each data point in Figure 4 represents combined data from two independent lines injected with the same concentration of DNA. The presence or absence of the transgene in mosaics was inferred by the presence of odr-1::dsRed, which is expressed in AWC and AWB neurons. The str-2::GFP expression phenotype in mosaic cells was examined under a compound scope at 100–400×. Previous experiments have used a similar strategy [9-12]. Statistical analysis was performed for mutant mosaics rescued at 2.5 or 5 ng/ul of odr-3:olrn-1b, and for wild-type mosaics injected with 15 or 25 ng/μl of odr-3:olrn-1b, to test: the null hypothesis that both AWC neurons behaved as independent units, purely as predicted by the proportions in the non-mosaic controls; the null hypothesis that the DsRed-positive (rescued) AWC behaved purely as predicted by the rescued controls; and the null hypothesis that the DsRed-negative (non-rescued) AWC behaved purely as predicted by the non-rescued controls. In all cases, results were different from the null hypothesis at P < 0.001 by Chi square test or Fisher exact test as appropriate, using the calculator at [42]. Both the rescued AWC and the non-rescued AWC in olrn-1; odr-3::olrn-1b mosaics became AWCON more often than predicted by the null hypothesis. Thus, there is both autonomous and non-autonomous rescue of olrn-1. In wild-type mosaics overexpressing olrn-1, the overexpressing AWC became AWCON more often than predicted by the null hypothesis, and the wild-type AWC became AWCOFF more often than predicted.

In rare olrn-1 mosaic animals, the transgene was lost in both AWC neurons but retained in either or both AWB neurons. These animals did not express str-2::GFP (n = 5 animals), suggesting that olrn-1 expression in AWC accounts for its major role in AWC asymmetry.

Mosaic analysis of olrn-1 in nsy-4 or nsy-5 mutants was performed on a single line per genetic background. A single unstable extrachromosomal array was examined in wild-type, nsy-4(ky616) or nsy-5(ky634) backgrounds bearing stable integrated str-2::GFP transgenes.

Loss-of-function mosaic analysis for unc-2 was performed on three independent lines with unstable extrachromosomal transgenic arrays [odr-3::unc-2, odr-1::dsRed] in a kyIs140 [str-2::GFP] I; unc-2(lj1) X mutant. Gain-of-function mosaic analysis for unc-2 was performed on three independent lines in the kyIs140 I strain. Loss-of-function mosaic analysis for unc-36 was performed on three independent lines with unstable extrachromosomal transgenic arrays [odr-3::unc-36-SL2-CFP, odr-1::dsRed] in a kyIs140 I; unc-36(e251) III mutant. Gain-of-function mosaic analysis for unc-36 was performed on three independent lines in the kyIs140 strain. Loss of the transgene was inferred by loss of the co-injection marker odr-1::dsRed in AWC neurons. Results from all lines were combined for statistical analysis, which was performed as described above for olrn-1. In all cases the null hypothesis that the two AWCs behaved as independent units could be excluded at P < 0.001. Both the rescued AWC and the non-rescued AWC in unc-2; odr-3::unc-2 mosaics were strongly affected by the contralateral cell – the rescued AWC became AWCOFF more often than predicted by the null hypothesis, and the non-rescued AWC became AWCON more often than predicted (P < 0.001 in both cases). Thus, a single unc-2(+) AWC can bias both AWC neurons. In wild-type mosaics overexpressing unc-2, the wild-type AWC became AWCOFF more often than predicted by the null hypothesis (P = 0.003), but the overexpressing AWC was not affected by the wild-type AWC (P = 0.1943). In unc-36; odr-3::unc-36 mosaics, the wild-type AWC became AWCOFF more often than predicted by the null hypothesis (P < 0.001), but the mutant AWC was not significantly affected (P = 0.075). There was no significant effect of unc-36 overexpression in a wild-type background.

Microscopy

To visualize early L1s, 50–100 gravid adults were picked and allowed to lay eggs overnight. Adults and hatched larvae were washed off the plate, and eggs were allowed to hatch for three hours. L1 larvae were examined on a compound microscope at 400–630× magnification or used for confocal microscopy. To obtain late L1/early L2 animals, approximately 50–60 gravid adults were picked to a plate and allowed to lay eggs for 3 hours, after which the adults were removed from the plate. Progeny were examined 30 hours later under the confocal microscope.

Competing interests

The author(s) declare that they have no competing interests.

Authors' contributions

SLBH, YS, and CIB designed the experiments, and SLBH and YS performed the experiments. MKV isolated the olrn-1(ky626) mutant, and IT, TI, and IK cloned olrn-1. AvdL and PS characterized the srsx-3 reporter. SLBH and CIB wrote the paper.

Acknowledgements

We thank Chiou-Fen Chuang, Bibi Lesch, David Sretavan, Steve McIntire, and Herwig Baier for their insights throughout this work, and Andy Chang for comments on the manuscript. This work was funded by NIH grants DC04089 to CIB and GM56223 to PS. CIB is an Investigator of the Howard Hughes Medical Institute.

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