The FLP Neurons Respond to Harsh Head Touch and Gentle Nose Touch
(A) Connections between nose touch mechanoreceptors. Shown are synaptic and electrical connections involving FLP and other nose touch mechanoreceptors. Gap junctions are indicated by dashed lines, chemical synapses by solid lines, with triangles signifying presynaptic terminals. Known or hypothesized outputs of sensory neurons (Alkema et al., 2005; Hart et al., 1995; Sawin et al., 2000) are indicated. Also indicated are sites of transgenic rescue for genes involved in nose touch behavior and nose-touch-evoked calcium transients in FLP as determined in this study. MEC-10 acts cell autonomously in the FLPs; OSM-9 acts in the OLQs, and TRP-4 acts in the CEPs and other dopaminergic mechanoreceptors. OSM-9 also acts in the ASH neurons to promote nose touch behavior, though expression here does not affect neural responses to nose touch in FLP (see Figure 3).
(B–D) Averaged calcium responses to harsh head touch (B), gentle head touch (C), and gentle nose touch (D). Each red trace represents the average percentage change in R/R₀ for the indicated genotype, where R is the fluorescence emission ratio at a given time point, and R₀ is its initial value. The number of individual recordings averaged for each stimulus condition was 24 (harsh head touch), 21 (gentle head touch), and 12 (gentle nose touch). Gray shading indicates SEM of the mean response. Scale bars are indicated.

MEC-10 Is Required Cell Autonomously for FLP Harsh Touch Response
Averaged responses of wild-type (A), mec-10(tm1552) (B), osm-9(ky10) (C), and mec-10(tm1552); egl-46::mec-10 (D) to harsh head touch stimulation in FLP. Each red trace represents the average percent change in R/R₀ for 21 (wild-type, osm-9, and mec-10; ljEx220[egl-46::mec-10genomic]) or 14 (mec-10) individual recordings. Gray shading indicates the SEM. None of these genotypes visibly altered the morphology of FLP, or the expression pattern of the cameleon transgene.
(E) Scatter plot of peak calcium responses for each genotype. Statistical significance (^∗∗∗p < 0.0005) is according to the Mann-Whitney U rank sum test. Also shown are data for mec-10(tm1552); egl-46::mec-10cDNA (n = 23), PVD-ablated mec-10 (n = 14) and mec-10(tm1552); egl-46::mec-10 (n = 9), and mock-ablated mec-10 (n = 11) and mec-10(tm1552); egl-46::mec-10 (n = 14). These results, together with those in Figure S3, demonstrate that the transgenic rescue results specifically from expression of mec-10(+) in FLP, and not PVD.

MEC-10 Is Required Cell Autonomously and OSM-9 Nonautonomously for FLP Nose Touch Response
(A) Averaged responses of wild-type, mec-10(tm1552), osm-9(ky10), and osm-9(ky10); mec-10(tm1552) to gentle nose touch stimulation in FLP. Each red trace represents the average percentage change in R/R₀ for wild-type (n = 24), mec-10 (n = 22), osm-9 (n = 22), and osm-9;mec-10 (n = 13) individual recordings; gray shading indicates SEM.
(B and C) Scatter plot of peak calcium responses for each genotype. In addition to the genotypes in (A), we analyzed mec-10(tm1552); egl-46::mec-10(genomic) (n = 13) and mec-10(tm1552); egl-46::mec-10(cDNA) (n = 13) rescue lines in (B), and osm-9(ky10) rescue lines expressing osm-9(+) under the del-2 (genomic fragment, n = 13), egl-46 (genomic fragment, n = 15; cDNA n = 10), sra-6 (genomic fragment, n = 17), or ocr-4 (genomic fragment, n = 10; cDNA, n = 9) promoters in (C). For (B), also shown are data for mec-10 mutant (n = 13) and rescue animals (n = 13) in which the PVD harsh body touch neurons have been eliminated by laser ablation; these results demonstrate that the transgenic rescue results specifically from expression of mec-10(+) in FLP.
(D and E) Effects of mec-10 and osm-9 on nose touch escape behavior. For all genotypes at least 50 animals were scored for reversals following nose touch stimulation. Statistical significance (^∗∗p < 0.001, ^∗∗∗p < 0.0001) is according to the Student's t test.

Effect of osm-9 on OLQ Nose Touch Responses
(A) OSM-9 is required cell autonomously for OLQ nose touch response. Shown are averaged responses of wild-type (n = 14), osm-9(ky10) (n = 10), and osm-9(ky10); ocr-4::osm-9(genomic) (n = 10) to nose touch stimulation in OLQ. Gray shading indicates the SEM. None of these genotypes visibly altered the morphology of OLQ, or the expression pattern of the cameleon transgene (see Figure S4).
(B) Scatter plot of peak OLQ calcium responses for osm-9 genotypes. In addition to the strains shown in (A), we imaged ten animals in which osm-9(ky10) was rescued by an ocr-4::osm-9 cDNA transgene.

Effects of OLQ and CEP Sensory Inputs on Nose Touch
(A and B) Effects of trp-4 and trpa-1 on FLP nose touch responses. As diagrammed in (A), we imaged FLP nose touch responses in animals carrying mutations affecting the OLQ (trpa-1) or CEP (trp-4) neurons. Shown in (B) is a scatter plot of peak calcium responses (percentage change in R/R₀) for 16 wild-type, 16 trp-4(ok1605), 16 trp-4; egl-46::trp-4, 16 trp-4; dat-1::trp-4, 16 osm-9(ky10); trp-4(ok1605), 11 trpa-1(ok999), 11 trpa-1; egl-46::trpa-1, 9 trpa-1; del-2::trpa-1, and 16 trpa-1; sra-6::trpa-1 animals. Statistical significance (^∗∗∗p < 0.0005) is according to the Mann-Whitney U rank sum test.
(C and D) Effect of the trp-4 and trpa-1 on nose touch behavior. For all genotypes at least 50 animals were scored for reversals following nose touch stimulation. Statistical significance (^∗∗p < 0.001, ^∗∗∗p < 0.0001) is according to the Student's t test.

The RIH Interneuron Integrates Responses to Nose Touch
(A) Averaged responses of wild-type, trp-4(ok1605) osm-9(ky10), and osm-9(ky10); trp-4(ok1605) to nose head touch stimulation in RIH. As diagrammed, we imaged RIH calcium transients in response to nose touch stimulation. Each solid trace represents the average percentage change in R/R₀ for 20 wild-type, 14 osm-9(ky10), 16 trp-4(ok1605), and 12 osm-9(ky10); trp-4(ok1605) individual animals. Gray shading indicates the SEM. None of these genotypes visibly altered the morphology of RIH (data not shown), or the expression pattern of the cameleon transgene (Figure S4).
(B) Scatter plot of peak calcium responses for each genotype. In addition to the genotypes in (A), ten unc-13, ten unc-7, 14 unc-7; egl-46::unc-7; cat-1::unc-7 ; 13 trpa-1, 16 trpa-1; ocr-4::trpa-1, 20 trp-4; dat-1:: trp-4, 13 mec-10, 16 mec-10; egl-46::mec-10(cDNA), 20 osm-9; ocr-4::osm-9(genomic), and ten osm-9; ocr-4::osm-9(cDNA) individual animals were analyzed. Statistical significance (^∗∗∗p < 0.0005) is according to the Mann-Whitney U rank sum test.

The RIH Network Is Important for FLP Responses to Nose Touch but Not Harsh Head Touch
(A) Imaging the effect of RIH ablation on FLP responses to nose touch and harsh head touch. As diagrammed, we recorded calcium transients in FLP following mechanical stimulation in intact and RIH-ablated animals.
(B) Responses of wild-type and RIH-ablated animals to nose touch stimulation. Each solid trace represents the average percentage change in R/R₀ for 24 (mock-ablated, red trace) or 13 (RIH-ablated, green trace) individual recordings. Gray shading indicates SEM of the mean response. Scale bars are indicated above. The green bar indicates the time of the stimulus. Ablation of RIH did not visibly alter the morphology of FLP or RIH, or the expression patterns of the cameleon transgenes. Scatter plot shows peak responses of 20 mock-ablated and 20 RIH-ablated animals. Statistical significance (^∗∗∗p < 0.0005) is according to the Mann-Whitney U rank sum test.
(C) Effect of RIH ablation on nose touch escape behavior. Animals were touched on the nose, and escape responses (reversals) were scored as described. At least 100 animals were tested for each genotype. Statistical significance (^∗∗∗p < 0.0005) is according to the Student's t test.
(D) Responses of wild-type and RIH-ablated animals to harsh head touch stimulation. Each solid trace represents the average percentage change in R/R₀ for 24 (mock-ablated, red trace) or 13 (RIH-ablated, green trace) individual recordings. Gray shading indicates SEM of the mean response. Scale bars are indicated above. Scatter plot shows peak responses of 20 mock-ablated animals, 20 RIH-ablated animals, and six unc-7 mutant animals (unc-7 nose touch responses are in Figure S7).

The RIH Network Is Important for OLQ Responses to Nose Touch and Harsh Head Touch
(A) Imaging OLQ responses to nose touch and harsh head touch. As diagrammed, we recorded calcium transients in OLQ following mechanical stimulation in wild-type, ablated, and mutant animals.
(B) Scatter plot of peak calcium responses to nose touch or harsh head touch in OLQ. For harsh head touch, 14 wild-type, ten RIH-ablated wild-type, seven mock-ablated wild-type, ten mec-10(tm1552), and ten mec-10(tm1552); egl-46::mec-10(cDNA) were imaged. For nose touch, 14 wild-type, ten RIH-ablated wild-type, seven mock-ablated wild-type, ten mec-10(tm1552), and ten mec-10(tm1552); egl-46::mec-10(cDNA) were imaged. Statistical significance (^∗∗∗p < 0.0005) is according to the Mann-Whitney U rank sum test.
(C) Calcium responses in OLQ to harsh head touch. Red traces indicate the average percentage change in R/R₀ for selected genotypes from (B). Gray shading indicates SEM. None of these genotypes visibly altered the morphology of OLQ, or the expression pattern of the cameleon transgene.
(D) Calcium responses in OLQ to nose touch. Red traces indicate the average percentage change in R/R₀ for selected genotypes from (B). Gray shading indicates SEM.