Interaxonal interaction defines tiled presynaptic innervation in C. elegans

Abstract

Cellular interactions between neighboring axons are essential for global topographic map formation. Here we show that axonal interactions also precisely instruct the location of synapses. Motoneurons form en passant synapses in Caenorhabditis elegans. Although axons from the same neuron class significantly overlap, each neuron innervates a unique and tiled segment of the muscle field by restricting its synapses to a distinct subaxonal domain-a phenomenon we term synaptic tiling. Using DA8 and DA9 motoneurons, we found that the synaptic tiling requires the PlexinA4 homolog, PLX-1, and two transmembrane semaphorins. In the plexin or semaphorin mutants, synaptic domains from both neurons expand and overlap with each other without guidance defects. In a semaphorin-dependent manner, PLX-1 is concentrated at the synapse-free axonal segment, delineating the tiling border. Furthermore, plexin inhibits presynapse formation by suppressing synaptic F-actin through its cytoplasmic GTPase-activating protein (GAP) domain. Hence, contact-dependent, intra-axonal plexin signaling specifies synaptic circuits by inhibiting synapse formation at the subcellular loci.

Figure 1. DA8/DA9 synaptic tiling depends on axon-axon interaction

(A) Schematic representative of the DA8 (green) and DA9 (magenta) neuron. Cell bodies are shown as big circles, and ovals represent synapses. Three parameters used in this study are shown. (B) Quantification of overlap between DA8 and DA9 synaptic domains in unc-13 and axon guidance mutants. Error bars; standard error of mean. Triple asterisks; p<0.001 n.s.; not significant (ANOVA/Dunnett). (C-G) representative images of the synaptic patterns in DA8 and DA9 of wildtype (C); unc-129 with DA8 misguided (D), DA9 misguided (E), or normal axon guidance (F); and unc-13 mutants (G). Arrows denote the tiling border. Overlap between the DA8 and DA9 synaptic domains are indicated in yellow bars. Scale bar, 20μm. See also Figure S1.

Figure 2. plexin and semaphorin mutants have synaptic tiling defects

(A-C) Synaptic tiling defect in L4 animals of wildtype (A) plx-1(nc36) (B) and smp-1;smp-2 (C) mutants. Overlap between the DA8 and DA9 synaptic domains are indicated in yellow bars. Magnified images (represented by dotted boxes) and line-scan images within the magnified region were also shown. (D) Schematic representation of the tiling mutant phenotype. (E) Quantification of overlap between DA8/DA9 synaptic domains. Error bars; standard error of mean. Triple asterisks; p<0.001, Single asterisk; p<0.05 n.s.: not significant (ANOVA/Tukey-HSD). (F) Schematic representation of the genomic locus of the plx-1 gene. White boxes represent exons. The grey bar represents the deletion in nc36. The res asterisk indicates the position of deletion found in wy592 allele. DNA and corresponding amino acid sequences around the deletion found in wy592 of wildtype and wy592 are shown in the boxes. Sequence deleted in wy592 allele is shown in red. See also Figure S1 to S4.

Figure 3. Both plx-1 and smp-1 function cell autonomously in DA9

(A) Cell specific rescue of plx-1 with PLX-1 cDNA and its mutant variants expressed from cell-specific promoters. DA-specific (Punc-4c) and DA9-specific (Pitr-1 and Pmig-13) PLX-1 expression rescued the tiling phenotype in the plx-1 mutant. (B) Cell-specific rescue of smp-1;smp-2 mutants by wild-type or mutant SMP-1 variants expressed from cell-specific promoters. DA-specific (Punc-4c) and DA9 specific (Pitr-1 and Pmig-13) SMP-1 expression rescued DA8/9 synaptic overlap. (C) Schematics of PLX-1, SMP-1 and their deletion mutants used in this study. PLX-1 has one SEMA domain (white circle), three PSI domians (grey circles) and three glycine-proline rich repeats (black boxes) in the extracellular domain. There is a conserved RasGAP domain (white boxes) in the cytoplasmic domain. Asterisk indicates the position of deletion found in wy592 allele. SMP-1 has one SEMA domain (white circle) and one PSI domain (grey circles). (D) Cell specific rescue of smp-1;smp-2;plx-1 mutants with PLX-1 and/or SMP-1 cDNA. Expression of both PLX-1 and SMP-1 cDNA from DA9 specific promoters (Pitr-1 and Pmig-13) rescued the triple mutants. Mosaic experiment using DA specific promoter (Punc-4c) was alsho shown. (−) indicates the loss of transgene in the cell. Triple asterisks: p<0.001, n.s.: not significant (ANOVA/Dunnett). Error bars; standard error of mean. See also Figure S5 to S7.

Figure 4. PLX-1 functions throughout development

(A and B) Synaptic tiling at early L1 stage of wild type (A) and plx-1 mutants (B). A tiling defect was observed at this stage in the plx-1 mutants (yellow arrow). Scale bar, 10μm. (C) Quantification of DA8/DA9 overlap at L4 stage. Two independent transgenic lines and their siblings without the transgenes were quantified. Heat shock induction of plx-1 gene expression from the hsp promoter at the embryonic (red bars) and larval (orange bars) stages significantly rescued the plx-1 mutant phenotype while there was no significant difference between transgenic animals and non-transgenic siblings without heat shock (white bars). Error bars; standard error of mean. Triple asterisks: p<0.001, n.s.: not significant (ANOVA/Tukey-HSD). (D) Schematic representation of worm developmental time course and the timing of heat shock and imaging.

Figure 5. PLX-1::GFP is enriched at a synapse-free domain adjacent to the synaptic region

PLX-1::GFP localization in wildtype (A, B), in unc-129 mutants with DA9 axon misguided (C, D) and in smp-1;smp-2 mutants (E, F). Each strain has a synaptic marker (Pmig-13::mCherry::rab-3) and merged images are shown in the right panel (B, D and F). (G) PLX-1(ΔSema)::GFP localization in wildtype. (H) PLX-1(ΔStalk)::GFP localization in wildtype. (I) Quantification of the normalized mCherry::RAB-3 signal (magenta) and PLX-1::GFP signal (green) in the dorsal axon of wildtype worms. 18 animals were aligned according to the PLX-1::GFP patch at the anterior edge of the DA9 synaptic domain (orange arrow). Light colors indicate standard error of mean. (J) PLX-1::GFP merged with mCherry::RAB-3 in unc-104/kinesin mutants. mCherry::RAB-3 signal is completely absent from the dorsal axon (K). PLX-1 at the putative tiling border is indicated by yellow arrows. (L) SMP-1::GFP localization in wildtype. (M) mCherry::RAB-3 co-labeled with SMP-1::GFP. Scale bar, 20μm.

Figure 6. PLX-1 tiles synapses by inactivating Ras in DA9

(A-D) Subcellular localization of PLX-1 lacking its cytoplasmic domain (ΔCyto) (A and B) and RasGAP-deficient PLX-1 (PLX-1(RA)::GFP) in the plx-1 mutants (C and D). PLX-1 signal at the putative tiling border is indicated by yellow arrows, and ectopic synapses (mCherry::RAB-3) in the anterior axon are indicated by white arrowheads in (Band D). (E) Quantification of the normalized mCherry::RAB-3signal (magenta) and PLX-1(RA)::GFP signal (green) in the dorsal axon of wildtype worms. 18 animals were aligned according to the PLX-1::GFP patch at the putative anterior edge of the DA9 synaptic domain (orange arrow). Black arrows indicate ectopic RAB-3 signal anterior to the PLX-1(RA) localization. The light-green and light-magenta traces indicate standard error of mean. Scale Bars, 20 μm. (F) Synaptic tiling defect in let-60 (Ras) gain-of-function mutants. Yellow bar indicates DA8/DA9 synaptic overlap. (G) Genetic interaction between plx-1 and let-60(gf), and cell-specific suppression of let-60(gf) by two dominant negative LET-60 mutants. Triple asterisks; p<0.001, Double asterisks; p<0.01, n.s.: not significant (ANOVA/Tukey-HSD). Scale bar, 20μm.

Figure 7. F-actin is enriched at the DA9 synaptic domain and is expanded in the synaptic tiling mutants

(A-H) GFP::Utrophin-CH pattern in DA9 of wildtype (A, B), plx-1 mutants (C, D), smp-1;smp-2 double mutants (E, F) and let-60(n1046gf) mutants (G, H), either GFP::Utrophin-CH signal alone (A, C, E and G), or overlaid with mCherry::RAB-3 (B, D, F and H). (I) Quantification of Utrophin-CH domains in the dorsal axon. The length of the Utrophin-CH domain from the most posterior-dorsal axon (indicated with white arrowheads) to the anterior edge (indicated with yellow arrows) was measured. Triple asterisks; p<0.001. Double asterisks; p<0.01, Single asterisk; p<0.05 (ANOVA/Tukey-HSD). Error bars; standard error of mean. (J) GFP::Utrophin-CH and mCherry::RAB-3 double labeling in unc-104 mutants. Note that the mCherry::RAB-3 signal is completely absent in the dorsal axon. Scale bar, 20μm.

Figure 8. Model of the synaptic tiling in DA8/DA9

PLX-1 is localized at the synaptic tiling border dependent on both SMP-1 in DA9 and unknown molecule(s) in DA8. Localized and activated PLX-1 inhibits synapse formation in DA9 as well as restricts the activity or localization of unknown molecule(s) in DA8. See text for detail.