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Fig. 1. Photoirradiation opens the pylorus in sea urchin larvae. a Photoirradiation drives the pyloric opening. Images of 5 s and 1 min 30 s after photoirradiation were captured from Supplementary Movie 1. b The set light condition was 10 min of light, overnight in darkness, and photoirradiation by high-intensity light. The graph shows the pyloric opening rates between 0 and 10 min after photoirradiation. The fluorescence images show pyloric closing (left) and opening (right), as visualized by TnI immunohistochemistry. N = 4; n (0 min) = 60, 39, 29, 43; n (1 min) = 45, 21, 30, 32; n (2 min) = 45, 26, 33, 54; n (3 min) = 43, 28, 23, 31; n (4 min) = 38, 28, 37, 24; n (5 min) = 68, 39, 35, 37; n (6 min) = 41, 28, 40, 26; n (7 min) = 72, 27, 23, 39; n (8 min) = 43, 27, 31, 13; n (9 min) = 84, 29, 29, 29; n (10 min) = 76, 38, 24, 21. The increase in the opening rate between 0 and 2 min after photoirradiation was assessed by the Welch two-sample t-test. Error bars show SE. More than 40% of larvae opened their pylorus in response to the photoirradiation (= sum of open rate from 1 to 5 min (8.9% (1 min) + 17.9% (2 min) + 16.1% (3 min) + 10.9% (4 min) + 7.0% (5 min)) was divided by 1.5 min (average open time)). c The region around the anterior neuroectoderm was necessary for pyloric opening induced by photoirradiation. The images in the upper row show the location at which the anterior neuroectoderm and pre-oral arms (− ANE and pre-oral arms) and post-oral arms (− post-oral arms) were removed. The lower images show open and closed pylori 2 min after photoirradiation. The inset of each image shows the bright-field images. The graph shows the opening rate of the pylorus in control larvae, larvae without the AP and pre-oral arms, and larvae without post-oral arms 2 min after photoirradiation. N = 2–4; n (control) = 19, 20, 24, 45; n (ANE and pre-oral arms less) = 13, 15, 17, 17; n (post-oral arms less) = 13, 12. We used one-way ANOVA followed by Tukey’s post hoc test. Error bars show SE. Scale bars in a and b = 20 μm and in c = 50 μm
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Fig. 2. The anterior neuroectoderm plays a crucial role in opening the pylorus. a Serotonergic neurons and pan-neurons recognized by Synaptotagmin B (synB) in sea urchin larvae. Green, serotonin; magenta, SynB; blue (DAPI), nuclei. b Serotonin induced pyloric opening in both control larvae and larvae without the ANE and pre-oral arms. The inset of each image shows the bright-field images. c The graph shows the opening rate of the pylorus in control larvae treated with seawater, in control larvae 2 min after the addition of serotonin, and in larvae without the ANE and pre-oral arms 2 min after the addition of serotonin. N = 3; n (with seawater) = 55, 79, 74; n (with serotonin) = 23, 11, 10; n (without the ANE and pre-oral arms and with serotonin) = 5, 8, 4. Error bars show SE. d The expression pattern of Go-Opsin (opn3.2) in Hemicentrotus pulcherrimus (arrows). e The activity of alkaline phosphatase in the gut was invariant in control and Go-Opsin morphants. f The graph shows the opening rate of the pylorus in control larvae and Go-Opsin morphants 2 min after photoirradiation or the addition of serotonin. N = 3; n (control; no treatment) = 45, 28, 22; n (control; photoirradiation) = 78, 63, 54; n (control; +serotonin) = 25, 49, 38; n (Go-Opsin morphants with no treatment) = 16, 14, 14; n (Go-Opsin morphants with photoirradiation) = 33, 32, 11; n (Go-Opsin morphants with serotonin) = 9, 51, 28. Error bars show SE. Scale bars in a, b, d, and e = 50 μm
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Fig. 3. The 5HT2 receptor mediates the light>pylorus signaling pathway. a The opening rate of the pylorus under photoirradiation was extremely reduced by the addition of melperone hydrochloride and ketanserin tartrate (5-HT2 receptor antagonists). N = 4; n (control) = 54, 26, 42, 66; n (melperone hydrochloride) = 15, 17, 27, 36; n (ketanserin tartrate) = 17, 17, 27, 25. Error bars show SE. b The putative cis-regulatory elements of the 5HT2 receptor drove Venus signaling in the stomach. The rate of Venus expression in the stomach was 81.4% (57/70) in all larvae that had Venus signals. s, stomach; i, intestine. c The activity of alkaline phosphatase in the gut was invariant in control and 5HT2 morphants. d The graph shows that the 5HT2 receptor was required for the light>pylorus signaling pathway. N = 2–3; n (control with no treatment) = 25, 37; n (control with photoirradiation) = 21, 11, 55; n (control with serotonin) = 25, 19, 22; n (control with SNAP) = 28, 14, 19; n (5HT2 morphants with no treatment) = 13, 16; n (5HT2 morphants with photoirradiation) = 16, 16, 17; n (5HT2 morphants with serotonin) = 35, 45, 23; n (5HT2 morphants with SNAP) = 28, 28, 37. Error bars show SE. Scale bars in b and c = 50 μm
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Fig. 4. The light>serotonin pathway activates enteric neurons in the stomach (sENs), which express nNOS and induce pyloric opening. a A schematic image of neuronal nitric oxide synthase (nNOS)-expressing enteric neurons (sENs; magenta) with axon-like structures around the pyloric sphincter (green) [23]. b The graph shows the pyloric opening rate in control and nNOS morphants under photoirradiation, the addition of serotonin, and the addition of SNAP. N = 3; n (control with no treatment) = 10, 21, 26; n (control with photoirradiation) = 14, 21, 28; n (control with serotonin) = 15, 12, 30; n (control with SNAP) = 21, 19, 13; n (nNOS morphants with no treatment) = 13, 11, 10; n (nNOS morphants with photoirradiation) = 21, 17, 20; n (nNOS morphants with serotonin) = 19, 26, 12; n (nNOS morphants with SNAP) = 63, 12, 29. Error bars show SE. c A schematic diagram of the light>pylorus signaling pathway in sea urchin larvae
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