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

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.

Figure 1

olrn-1 mutants have two AWC^OFF 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 AWC^OFF sensed odorant 2,3-pentanedione (pd) and the AWC^ON 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.

Figure 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.

Figure 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)).

Figure 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.

Figure 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 AWC^ON 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.

Figure 6

Model for calcium channel function and OLRN-1 in the AWC^ON/AWC^OFFdecision. 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 AWC^OFF transmits a signal to AWC^ON via NSY-5 gap junctions between AWC and other cells and NSY-4 claudins. This signal might be membrane potential. In AWC^ON, 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 AWC^ON. A feedback signal from the calcium channels and OLRN-1 is transmitted from AWC^ON back to AWC^OFF.