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. 2023 Jan 23;12:e76863. doi: 10.7554/eLife.76863

Figure 2. Anoctamins function in class III (CIII) neurons.

(A) For cold plate assay, larvae were acclimated to a room temperature arena before being transferred to a pre-chilled cold plate. Contraction (CT) was identified by measuring the length of skeletonized larvae over the course of chilling. (B) Mutant analysis; mean peak magnitude in larval CT. w1118 (n=30); subduedMi/+ (n=30; p=0.58; BF10=1.18); subdueddf/+ (n=30; p=1; BF10=0.28); subduedMi/Mi (n=30; p<0.001; BF10=228.63); subduedMi/df (n=30; p=0.008; BF10=737.03); wwkMi/+ (n=30; p=0.58; BF10=1.60); wwkdf/+ (n=30; p=0.003; BF10=324.11); wwkMi/Mi (n=30; p<0.001; BF10=433.18). Also see Figures 1—3. (C) Mutant analysis; % of animals which strongly CT in response to noxious cold (≥30% reduction in body length). Mutations in subdued and white walker result in a reduced percent of larvae which strongly CT in response to noxious cold (5°C). w1118 (n=30); subduedMi/+ (n=30; p=0.13; BF10=4.417); subdueddf/+ (n=30; p=0.25; BF10=2.826); subduedMi/Mi (n=30; p<0.001; BF10=461.34); subduedMi/df (n=30; p<0.001; BF10=997.24); wwkMi/+ (n=30; p=0.45; BF10=2.00); wwkdf/+ (n=30; p<0.001; BF10=997.24); wwkMi/Mi (n=30; p<0.001; BF10=997.24). (E) CIII-specific knockdown and rescue analyses; mean peak magnitude in larval CT. Knockdown: GAL4 control (n=30); subdued RNAi-1 (n=30; p=0.049; BF10=3.98); subdued RNAi-2 (n=27; p=0.437; BF10=0.78); wwk RNAi-1 (n=30; p=0.005; BF10=89.25); wwk RNAi-2 (n=30; p<0.001; BF10=6932.18). Rescue: GAL4nompC;subduedMi/Mi (mutant, n=30); GAL4nompC/UAS-subdued;subduedMi/Mi (CIII rescue, n=30; p=0.049; BF10=1.61). wwkMi/Mi;GAL419-12 (mutant, n=30); wwkMi/Mi;GAL419-12/UAS-wwk (CIII rescue, n=30; p<0.001; BF10=83.59). (D) CIII-specific knockdown and rescue analyses; % of animals which strongly CT in response to noxious cold (≥30% reduction in body length). CIII-specific knockdown (GAL419-12) of subdued and white walker results in a reduced percent of larvae which strongly CT in response to noxious cold. GAL4 control (n=30); subdued RNAi-1 (n=30; p=0.039; BF10=7.64); subdued RNAi-2 (n=27; p=0.075; BF10=4.93); wwk RNAi-1 (n=30; p=0.002; BF10=71.17); and wwk RNAi-2 (n=30; p<0.001; BF10=138.15). GAL4-UAS-mediated CIII rescue of subdued and white walker in mutant backgrounds increased cold sensitivity. GAL4nompC;subduedMi/Mi (n=30); GAL4nompC/UAS-subdued;subduedMi/Mi (n=30; p=0.031; BF10=3.57). wwkMi/Mi;GAL419-12 (n=30); wwkMi/Mi;GAL419-12/UAS-wwk (n=30; p<0.001; BF10=281.95).

Figure 2.

Figure 2—figure supplement 1. Validation of mutagenic effects of subdued and white walker MiMIC mutations.

Figure 2—figure supplement 1.

(A) RT-PCR validating the subdued mutant. (B) RT-PCR validating the white walker (wwk) mutant. Relative to controls, which produced amplicons of the predicted sizes (see Materials and methods), homozygous Minos-mediated integration cassette (MiMIC) insertions for subdued or wwk led to the absence or severe reduction in amplicon products suggesting that these mutations are severely hypomorphic for subdued or wwk expression.
Figure 2—figure supplement 2. Cold nociception defects of subdued and white walker mutations.

Figure 2—figure supplement 2.

(A) Heatmap representation of cold-evoked contraction (CT) in genetic control (w1118) and mutant conditions, where FP is flaccid paralysis (full body relaxation with an inability to locomote). N=240, n=30 for each condition. (B) % of animals from anoctamin allele/deficiency experiments performing a particular CT response, where NR is no response (<10% reduction in length), weak is ≥10% reduction in length, moderate is ≥20% reduction in length, and strong is ≥30% reduction in length; bars show proportions in % ± standard error of the proportion (SEP). (C) % ± SEP of animals which ceased any contractile behavior whatsoever (returned to normal length) within the experimental time frame.
Figure 2—figure supplement 3. Cold nociception defects of subdued and white walker RNAi in CIII cold nociceptive neurons.

Figure 2—figure supplement 3.

(A) Heatmap representation of cold-evoked contraction (CT) in genetic control (GAL4) and knockdown conditions, where FP is flaccid paralysis. N=147, n=30 for each condition except subdued RNAi-2, where n=27. (B) % of animals from anoctamin knockdown experiments performing a particular CT response, where NR is no response (<10% reduction in length), weak is ≥10% reduction in length, moderate is ≥20% reduction in length, and strong is ≥30% reduction in length; bars show proportions in % ± standard error of the proportion (SEP). (C) % ± SEP of animals which ceased any contractile behavior whatsoever (returned to normal length) within the experimental time frame.
Figure 2—figure supplement 4. Rescue analyses of cold nociceptive defects for subdued and white walker.

Figure 2—figure supplement 4.

(A) Heatmap representation of cold-evoked contraction (CT) in mutant GAL4 control and GAL4-UAS-mediated rescue conditions, where FP is flaccid paralysis. N=120, n=30 for each condition. (B) % of animals from rescue experiments performing a particular CT response, where NR is no response (<10% reduction in length), weak is ≥10% reduction in length, moderate is ≥20% reduction in length, and strong is ≥30% reduction in length; bars show proportions in % ± standard error of the proportion (SEP). (C) % ± SEP of animals which ceased any contractile behavior whatsoever (returned to normal length) within the experimental time frame.