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. 2021 Aug 5;10:e66144. doi: 10.7554/eLife.66144

Figure 6. r-opsin1 mediates blue light modulation of trunk r-opsin1-expressing (TRE) signature and undulation behavior.

(A) atp2b/c7424 expression levels (in transcripts per million reads [TPMs]) in individual replicates of r-opsin1+/+ and r-opsin1-/- worms cultured for 3–5 days in bright blue light. For Atp2b2 phylogeny, see Figure 6—figure supplement 1. (B) Scheme of decapitated worm trunks as used in experiments (D–H) that survive for up to 14 days. (C) Spectral profile of bright blue and dim white light. The blue dot indicates the irradiance at 471 nm (λmax of P. dumerilii r-Opsin1). (D) atp2b/c7424 expression levels (in TPMs) in individual replicates of decapitated worms cultured for 3–5 days in bright blue light or dim white light. (E–H) Undulation behavior of decapitated worms. (E) Light regime. Black portions of the horizontal bar indicate ‘night’ (light off), yellow portions indicate ‘day’ (light on), and gray portions indicate ‘subjective day’ (light off during ‘day’ period). ZT: zeitgeber time; CT: circadian time. (F, G) Undulation ratio during ‘day’ (F) and ‘subjective day’ (G). Each black (red) point represents the mean of all wild-type (mutant) worms within a 3 min window, and vertical bars represent the standard error of the mean (n = 32 for each genotype, distributed among three independent experiments). For reliability tests of the algorithm used to detect undulation behavior, see Figure 6—figure supplement 3. (H) Area under the curve obtained from the undulation ratios shown in (F) (yellow background; ‘Day’) and (G) (gray background; ‘Subjective day’). Circles indicate data corresponding to individual worms. Boxplots indicate the median (thick horizontal line), the 50% quantile (box), and 100% quantile (error bars). Filled circle indicates an outlier (as determined by the boxplot function of the ggplot R package). *p-value<0.05; **p-value<0.01 (Wilcoxon rank-sum and signed-rank tests). For behavioral responses to strong light of r-opsin+/+ and r-opsin-/- trunks, see Figure 6—figure supplement 2.

Figure 6.

Figure 6—figure supplement 1. Atp2b2 phylogeny and enrichment of atp2b2 in zebrafish neuromasts.

Figure 6—figure supplement 1.

(A) Phylogenetic tree including Atp2b2 and related genes. Mm: Mus musculus; Dr: Danio rerio; Dm: Drosophila melanogaster; Ce: Caenorhabditis elegans; Pdu: Platynereis dumerilii; Nv: Nematostella vectensis; Aq: Amphimedon queenslandica. Red clade: Atp2b protein family; green clade: Atp2a protein family; blue clade: Atp2c protein family. Arrowheads indicate proteins encoded by genes mentioned in the article: black: mouse Atp2b2; dark blue: zebrafish Atp2b2; Violet: Drosophila Pmca; green: C. elegans Mca-3; red: Platynereis Atp2b. For sequence identifiers see Supplementary file 3. (B) Bar plots indicate fold enrichment of actb, opn4xb, and atp2b2 mRNA expression (measured by quantitative PCR) in neuromast cells (FAC-sorted from fish trunks) as compared to unsorted cells from the same fish trunks. Enrichment values were normalized to actb levels. As opn4xb (previously known as opn4x2) has been shown to be specifically expressed in the neuromasts of the lateral line within the trunk of the fish (Backfisch et al., 2013), the enrichment of opn4xb confirms the correct isolation of neuromasts cells. *p-value<0.05 (Student’s t-test).
Figure 6—figure supplement 2. Net avoidance crawling distance of decapitated r-opsin+/+ and r-opsin-/- worms in response to strong light.

Figure 6—figure supplement 2.

Dot plot showing the distance moved (in mm) by worms exposed to a bright light pulse (‘Light’) or not exposed (‘Dark’). *p-value<0.05 (Wilcoxon rank-sum test).
Figure 6—figure supplement 3. Benchmarking the algorithm used to detect undulation behavior.

Figure 6—figure supplement 3.

(A) Behavior arena of an individual worm showing the key points detected by the automatized tracker along the worm: jaws (‘jaws’), five points along the trunk (‘body1–5’), and tail (‘tail’). (B, C) Undulation ratio of wild-type (black) and mutant (red) worms as determined by a human observer (B) or by the automatized tracker (C). Each point represents the mean of all wild-type or mutant worms within a 3 min window, and vertical bars represent the standard error of the mean (n = 4 for each genotype).