Files in this Data Supplement:
Table S1. List of alleles used in this study.
Table S2. Primer sequences for verification of mutations in genetic crosses.
Table S3. List of transgenes used in this study together with primer sequences used to synthesize the plasmids.
Table S4. Microarray results detect Wnt receptors that are expressed in A-class motor neurons and negatively regulated by the UNC-4 pathway. Microarray experiments were performed with transcripts isolated from embryonic DA motor neurons and from larval VA motor neurons. Transcripts enriched in wild-type DA (lin-17=1.7×) or VA motor neurons (mig-1=1.6×) were identified by comparison to corresponding reference profiles of all cells. ‘EG’ (expressed gene) denotes transcripts that are detected but not enriched (Fox et al., 2005; Von Stetina et al., 2007a). promoter::GFP reporter genes for mom-5, mig-1 and lin-17 were detected in VA motor neurons (Fox et al., 2005) but expression or GFP reporters for cfz-2, lin-18 and cam-1 has not been reported (?). mom-5 and mig-1 transcripts are also upregulated (1.8×) in microarray profiles of unc-37 mutant VA motor neurons suggesting that the UNC-4 pathway negatively regulates mom-5 and mig-1 expression (Von Stetina et al., 2007b).
Table S5. Compilation of tapping assay data. Percentages of Unc versus non-Unc animals in each genetic background tested are listed. n≥50 for each genotype tested.
Table S6. Compilation of data of ceh-12::GFP expression in VA motor neurons. Percentages of VA motor neurons with ceh-12::GFP expression vs VAs with no ceh-12::GFP expression are listed. n≥10 for each VA scored; sum of pooled VA motor neurons scored is listed.
Table S7. Percentage of VA motor neurons with ectopic AVB to VA UNC-7S::GFP positive gap junctions vs percentage of cells with no UNC-7S::GFP. n≥10 for each VA scored; sum of pooled VA motor neurons scored is listed. These values were used to construct pie charts in supplementary material Figure S8.
Fig. S1. Wnt components differentially regulate ecoptic ceh-12::GFP expression in unc-4 mutant VA motor neurons. Apparent effects of cwn-2 (red) on ectopic ceh-12::GFP expression are not statistically significant. cfz-2 (green) is required for ectopic ceh-12::GFP expression in VA9 and lin-18 (purple) promotes ectopic ceh-12::GFP expression in VA8 and VA9. *P<0.05, **P<0.01, ***P< 0.0001 vs unc-4, Fisher’s Exact Test. n≥15 for each neuron.
Fig. S2. Components of the LIN-44-mediated pathway are not required for ceh-12::GFP expression in posterior VAs. Single and double mutants of lin-44 and cwn-1 and lin-17 do not significantly affect ectopic ceh-12::GFP expression in posterior VA motor neurons. Data from VA7-VA10 are pooled and the percentage of ceh-12::GFP positive and negative neurons is indicated. n≥15 for each neuron.
Fig. S3. Ectopic expression of EGL-20 is not sufficient to induce ceh-12 expression in WT VA motor neurons. (A) Confocal image of a wild-type (WT) worm expressing plim-4::EGL-20 and ceh-12::GFP. (B) Anterior ectopic EGL-20 expression (plim-4::EGL-20) does not result in ectopic ceh-12::GFP expression in a WT background (red). Anteriorly expressed EGL-20 results in ecoptic ceh-12::GFP expression in unc-4 mutant VAs throughout the length of the ventral nerve cord (black). n≥10 for each neuron.
Fig. S4. EGL-20/Wnt regulates the specificity of synaptic inputs to VA motor neurons. (A) RNAi of egl-20 in the hypormorphic unc-4(e2323) allele suppresses the Unc-4 backward movement defect. **P<0.01 vs unc-4, Fisher’s Exact Test, n=200. (B) Movement of ceh-12(gk391); unc-4(e2320); eri-1(mg366); lin-15b(n744) worms is not significantly different when treated with egl-20/Wnt RNAi compared with treatment with the empty vector control, n=200 (Winnier et al., 1999). (C) The deletion allele ceh-12(gk391) suppresses the Unc-4 backward movement defect of unc-4(e120) whereas the egl-20(hu120) null allele does not suppress unc-4(e120) and is partially epistatic to ceh-12(gk391). egl-20(hu120) alone does not show a backward movement defect (data not shown). Black brackets indicate significance of P<0.001 between connected pairs. N.S., not significant. n≥50. (D) Ectopic AVB gap junctions with VA7 and VA9 in unc-4(e120) are suppressed by egl-20(hu120). *P<0.05, **P<0.01. Taken together, these results indicate that EGL-20 may have separate threshold-dependent functions with competing outcomes: High levels of EGL-20 function are required for the creation of AVB to VA gap junctions and a minimum level of EGL-20 activity is necessary for the restoration of functional backward locomotory inputs to VAs in an unc-4 mutant. Hence, both hypomorphic and null alleles of egl-20 suppress the creation of ectopic AVB to VA gap junctions in unc-4 (see Fig. 6D) whereas Unc-4 suppression is conferred by hypomorphic egl-20 mutations (Fig. 3D, E; supplementary material Table S5) but not by the egl-20(hu120) null allele. n≥10 for each neuron.
Fig. S5. Mutations in Wnt receptors, cfz-2 and lin-18 do not affect Unc-4 movement. Movement of unc-4; lin-18 and unc-4; cfz-2 double mutants is not significantly different (P>0.05) from unc-4 single mutants. Fisher’s Exact Test. n≥50.
Fig. S6. RNAi of mom-5 in an unc-4 RNAi-sensitive strain suppresses the Unc-4 movement defect. Movement of unc-4(e2323); eri-1(mg366); lin-15b(n744) worms is significantly suppressed when treated with mom-5 RNAi compared with treatment with the empty vector control. ***P<0.0001 Fisher’s Exact Test. n≥50.
Fig. S7. Microarray analysis detects transcripts regulated by the unc-4 pathway in VA motor neurons. Microarray profiles of unc-37 mutant vs wildtype VA motor neurons were compared to detect differentially expressed transcripts (Von Stetina et al., 2007b). Fold change (x-axis) is plotted on a log2 scale against a significance score (y-axis). Thresholds for identifying transcripts with significantly different levels in unc-37 vs wildtype VAs were ≤5% FDR (false discovery rate) (horizontal dashed line) and fold change ≥ ±1.7× (vertical dashed lines). Blue diamonds correspond to transcripts downregulated in unc-37 mutant VAs and red diamonds represent transcripts that are upregulated in unc-37 vs. WT VA motor neurons. Gray diamonds represent transcripts with no significant fold change difference between unc-37 mutant and WT VAs. Enrichment in unc-37 VA motor neurons for selected genes ceh-12 (black diamond) (1.9×) and mig-1 (yellow diamond) (1.8×) and mom-5 (green diamond) (1.8×).
Fig. S8. Components of the LIN-17 pathway prevent the formation of VB-type inputs in VAs. (A) ‘Anterior’ VAs (VA2-6) show significantly more ectopic AVB to VA gap junctions in lin-17 and cwn-1 mutants vs WT. (B) No difference is detected in ectopic AVB to VA gap junctions in ‘posterior’ VAs (VA7-10). **P<0.01 *P<0.05, Fisher’s Exact Test vs WT. n≥10 for each neuron.
Fig. S9. LIN-44/Wnt antagonizes EGL-20-dependent expression of ceh-12::GFP in unc-4 mutant VA motor neurons. The double mutant lin-44; unc-4 has a similar effect on posterior ceh-12 expression as the unc-4 single mutant. EGL-20/Wnt is required for posterior ceh-12::GFP expression in unc-4 mutants. Ectopic ceh-12::GFP expression is partially restored in lin-44; unc-4; egl-20 triple mutants thereby demonstrating that lin-44 antagonizes ceh-12::GFP expression in posterior VAs. The occurrence of significantly more posterior ceh-12::GFP expression in lin-44; unc-4; egl-20 compared with unc-4; egl-20 also indicates that another pathway is promoting ceh-12 expression in these neurons in the absence of EGL-20/Wnt and LIN-44/Wnt activity. ***P< 0.001, Fisher’s Exact Test. Posterior VA motor neurons are grouped VA7-10. n≥15 for each neuron.
Fig. S10. BAR-1/β-catenin differentially affects movement in unc-37 and unc-4 mutants. (A,B) Mutation of bar-1 suppresses the Unc-37 backward movement defect (A) but not Unc-4 (P>0.05) (B). Movement was assessed with the tapping assay. n≥40. **P<0.01, Fisher’s Exact Test.
Fig. S11. The Wnt inhibitor pyrvinium suppresses unc-4(e2322ts) movement defect at 23°C. A tapping assay was performed to determine whether the canonical Wnt inhibitor pyrvinium was capable of suppressing the backword movement defect in a weak allele of unc-4. Pyrvinium showed a dose-dependent effect on suppression of Unc-4 movement. *P<0.05, **P≤0.001, ***P<0.0001, Fisher’s Exact Test. n≥100.
