Yaguchi J et al. (2024), Light-modulated neural control of sphincter reg...

ECB-ART-53328

Nat Commun 2024 Oct 18;151:8881. doi: 10.1038/s41467-024-53203-7.

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Light-modulated neural control of sphincter regulation in the evolution of through-gut.

Yaguchi J , Sakai K , Horiuchi A , Yamamoto T , Yamashita T , Yaguchi S .

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The development of a continuous digestive tract, or through-gut, represents a key milestone in bilaterian evolution. However, the regulatory mechanisms in ancient bilaterians (urbilaterians) are not well understood. Our study, using larval sea urchins as a model, reveals a sophisticated system that prevents the simultaneous opening of the pylorus and anus, entry and exit points of the gut. This regulation is influenced by external light, with blue light affecting the pylorus via serotonergic neurons and both blue and longer wavelengths controlling the anus through cholinergic and dopaminergic neurons. These findings provide new insights into the neural orchestration of sphincter control in a simplified through-gut, which includes the esophagus, stomach, and intestine. Here, we propose that the emergence of the earliest urbilaterian through-gut was accompanied by the evolution of neural systems regulating sphincters in response to light, shedding light on the functional regulation of primordial digestive systems.

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	Fig. 1. Light-induced anus opening in sea urchin larvae.a Lateral view of a sea urchin larva with the digestive tract illuminated, showing key anatomical features: the cardiac sphincter (, asterisk), pyloric sphincter (yellow), and anal sphincter (green). The inset on the bottom left depicts the anus in a closed state (arrowhead), while the bottom right shows the anus open and the intestine (i) contracted, 30 seconds post-photoirradiation. Annotations: M, mouth; e, esophagus; s, stomach; i, intestine. b Anus opening rates post-photoirradiation, assessed using anti-Troponin I (TnI) antibody staining (refer to Methods). The graph presents basic photoirradiation conditions alongside opening rates, with pyloric (blue) and anal (black) openings (N = 4 batches [each consisting of a different male and female pair], n [0 min] = 27, 32, 36, 46 larvae, n [1 min] = 18, 23, 43, 33 larvae, n [2 min] = 18, 37, 30, 45 larvae, n [3 min] = 14, 24, 42, 49 larvae, n [4 min] = 15, 11, 34, 39 larvae, n [5 min] = 21, 20, 40, 42 larvae, n [6 min] = 21, 25, 45, 36 larvae, n [7 min] = 24, 26, 27, 30 larvae, n [8 min] = 16, 27, 44, 40 larvae, n [9 min] = 20, 24, 52, 45 larvae, n [10 min] = 19, 25, 41, 44 larvae; pylorus: [0 min] mean 1.6% ± 1.6% SEM, [1 min] mean 11.1% ± 4.7% SEM, [2 min] mean 18.9% ± 1.2% SEM, [3 min] mean 10.8% ± 2.1% SEM, [4 min] mean 5% ± 1.9% SEM, [5 min] mean 4.8% ± 1.9% SEM, [6 min] mean 5.4% ± 2.3% SEM, [7 min] mean 3.8% ± 0.2% SEM, [8 min] mean 0.6% ± 0.6% SEM, [9 min] mean 0.6% ± 0.6% SEM, [10 min] mean 0.6% ± 0.6% SEM; anus: [0 min] mean 8.2% ± 2.2% SEM, [1 min] mean 31% ± 11% SEM, [2 min] mean 49.7% ± 8.9% SEM, [3 min] mean 46.3% ± 5.4% SEM, [4 min] mean 30.6% ± 4.4% SEM, [5 min] mean 34.8% ± 6.9% SEM, [6 min] mean 18.4% ± 3.7% SEM, [7 min] mean 16.3% ± 3.5% SEM, [8 min] mean 27.7% ± 11.2% SEM, [9 min] mean 14.5% ± 4.8% SEM, [10 min] mean 24.8% ± 4.1% SEM). c. Schematic of the experimental setup brain and pre-oral arm ablation with a needle. Color coding: orange, brain; blue, Opsin3.2-expressing cells; magenta, Opsin2-expressing cells; yellow, pylorus; green, anus. The graph illustrates the opening rates of the pylorus and anus 2 minutes after photoirradiation (N = 5 batches, n [control] = 95, 38, 54, 49, 37 larvae, n [brain & pre-oral arms removed] = 54, 22, 27, 28, 25 larvae; pylorus: [control] mean 17.2% ± 4.1% SEM, [brain & pre-oral arms removed] mean 3.5% ± 2.1% SEM; anus: [control] mean 40.7% ± 2.5% SEM, [brain & pre-oral arms removed] mean 58.8% ± 5.7% SEM). d, e Graphs depicting the opening rates of the pylorus and anus under various conditions: d serotonin treatment (10 µM) without photoirradiation (N = 4 batches, n [serotonin -] = 53, 42, 49, 53 larvae, n [serotonin +] = 35, 48, 45, 58 larvae; pylorus: [serotonin -] mean 4.3% ± 2% SEM, [serotonin +] mean 82.5% ± 7.7% SEM; anus: [serotonin -] mean 3.7% ± 1.2% SEM, [serotonin +] mean 5.5% ± 1.7% SEM); e serotonin treatment (10 µM) with photoirradiation (N = 3 batches, n [serotonin -] = 147, 71, 94 larvae, n [serotonin +] = 53, 31, 34 larvae; pylorus: [serotonin -] mean 20.4% ± 3.8% SEM, [serotonin +] mean 87.6% ± 11.5% SEM; anus: [serotonin -] mean 36.5% ± 5.8% SEM, [serotonin +] mean 2.6% ± 1.7% SEM). Statistical significance denoted as p < 0.05, **p < 0.01; n.s. = not significant, Welch’s t test (two-sided). Error bars shown in all graphs indicate SEM. Scale bar in a = 50 µm and in b =20 µm. Source data are provided as a Source Data file.
	Fig. 2. Light-induced anus opening is mediated by Opsin2.a Opsin2 cells are missing in Opsin2 morphants. Anus opening or closing is identified with anti-TroponinI (TnI) antibody (arrowhead). b–d Graphs depicting the opening rates of the pylorus and anus under various conditions: b in control versus Opsin2 morphants post-photoirradiation (N = 4 batches [each consisting of a different male and female pair], n [control] = 38, 77, 53, 19 larvae, n [Opsin2 MO-1] = 16, 10, 10, 20 larvae; pylorus: [control] mean 20.3% ± 5.4% SEM, [Opsin2 MO-1] mean 28.8% ± 4.3% SEM; anus: [control] mean 23.5% ± 4.5% SEM, [Opsin2 MO-1] mean 5% ± 5% SEM); c with or without atropine treatment (100 µM) (N = 3 batches, n [atropine -] = 57, 118, 61 larvae, n [atropine +] = 18, 42, 31 larvae; pylorus: [atropine -] mean 5% ± 2.5% SEM, [atropine +] mean 2.9% ± 1.6% SEM; anus: [atropine -] mean 6.2% ± 1.6% SEM, [atropine +] mean 34.1% ± 0.7% SEM); d with or without acetylcholine treatment (1 µM) with photoirradiation (N = 7 batches, n [acetylcholine -] = 38, 104, 70, 115, 114, 92, 147 larvae, n [acetylcholine +] = 46, 23, 40, 36, 33, 25, 62 larvae; pylorus: [acetylcholine -] mean 19.7% ± 4.3% SEM, [acetylcholine +] mean 29.2% ± 4.6% SEM; anus: [acetylcholine -] mean 39.5% ± 1.6% SEM, [acetylcholine +] mean 8% ± 3.6% SEM). e Illustration of the signaling pathways from light perception to pyloric and anal opening, mediated by Opsin proteins and neurotransmitters. Statistical significance denoted as p < 0.05, **p < 0.001; n.s. = not significant, Welch’s t test (two-sided). Error bars shown in all graphs indicate SEM. Scale bar in a =20 µm. Source data are provided as a Source Data file.
	Fig. 3. ChAT-positive anal neurons in sea urchin larvae.a The graph illustrates the number of anal neurons over time, showing the presence of approximately two neurons at the anus starting three days post-fertilization. (N = 3 batches [each consisting of a different male and female pair], n [2-day] = 4, 4, 3 larvae, n [3-day] = 6, 5, 6 larvae, n [4-day] = 8, 13, 3 larvae, n [5-day] = 4, 7, 3 larvae, n [6-day] = 8, 10, 10 larvae, n [7-day] = 7, 10, 8 larvae; [2 day] mean 0% ± 0% SEM, [3 day] mean 1.4% ± 0.1% SEM, [4 day] mean 1.9% ± 0.1% SEM, [5 day] mean 2% ± 0% SEM, [6 day] mean 1.9% ± 0.2% SEM, [7 day] mean 2.1% ± 0.1% SEM). b Immunostaining images of anal and intestine (yellow dotted-line) region with anti-SynB antibody (neurons) and anti-TroponinI (TnI) antibody (muscles). A rectangle indicated the magnified region shown in the right. The asterisks in the magnified view highlight the neurons. The schematic diagram presents a lateral view of the stomach and intestine. Magenta cells and green areas indicate neurons and sphincters, respectively, at anus. c–f Distribution of cholinergic neurons within the sea urchin larval intestine. d ChAT-neurons are not present around the pylorus. e Asterisks highlight neurons surrounding the anus. f Magenta cells in the schematic image depict ChAT-positive anal neurons. Scale bar in b =10 µm; c =20 µm. Source data are provided as a Source Data file.
	Fig. 4. TH-positive pyloric and anal neurons in sea urchin larvae.a The expression pattern of tyrosine hydroxylase (TH) mRNA in 60-hour larvae. TH is a rate-limiting enzyme for dopamine biosynthesis, with dopamine neurons present at the ciliary band, pylorus (arrowheads), and anus (asterisks). Magenta cells in the schematic image represent dopamine neurons at the pylorus and anus. b Arrows indicate stomach-side enteric neurons (sEN); arrowheads point to intestine-side enteric neurons (iEN); asterisks highlight neurons surrounding the anus. c Anal neurons (asterisks) co-express ChAT and TH. d Schematic representation of the sea urchin larval stomach and intestine, emphasizing neurons near the pylorus (yellow) and anus (green). Neuron types are color-coded: sEN (orange) as TH-/ChAT-, iEN (blue) as TH + /ChAT-, and perianal neurons (magenta) as TH + /ChAT + . Scale bar in a, b, and c =20 µm. Source data for micrograph are provided as a Source Data file.
	Fig. 5. Interactions between pyloric and anal opening pathways in sea urchin larvae.a Opening rates of the pylorus and anus in larvae treated with or without dopamine (0.1 µM) with photoirradiation (N = 4 batches [each consisting of a different male and female pair], n [dopamine -] = 114, 72, 71, 94 larvae, n [dopamine +] = 40, 29, 33, 53 larvae; pylorus: [dopamine -] mean 30.5% ± 4% SEM, [dopamine +] mean 36% ± 4.8% SEM; anus: [dopamine -] mean 35.8% ± 4% SEM, [dopamine +] mean 7.8% ± 0.5% SEM). b Opening rates of the pylorus and anus in larvae treated with or without the dopamine inhibitor amisulpride (50 µM) (N = 5 batches, n [amisulpride -] = 38, 67, 40, 47, 46 larvae, n [amisulpride +] = 20, 55, 48, 41 36 larvae; pylorus: [amisulpride -] mean 6.2% ± 1.6% SEM, [amisulpride +] mean 2.6% ± 2.2% SEM; anus: [amisulpride -] mean 5.2% ± 1.2% SEM, [amisulpride +] mean 18.4% ± 2.9% SEM). c Opening rates of the pylorus and anus in larvae exposed to a combination of inhibitors (100 µM atropine or 50 µM amisulpride) and serotonin (10 µM; applied 5 min after inhibitor treatment), illustrating the modulatory effects on opening behavior (N = 3 batches, n [atropine -, amisulpride -, serotonin -] = 27, 21, 30 larvae, n [atropine -, amisulpride -, serotonin +] = 27, 31, 31 larvae, n [atropine +, amisulpride -, serotonin -] = 20, 20, 23 larvae, n [atropine +, amisulpride -, serotonin +] = 19, 31, 24 larvae, n [atropine -, amisulpride +, serotonin -] = 21, 10, 26 larvae, n [atropine -, amisulpride +, serotonin +] = 23, 30, 17 larvae; pylorus: [atropine -, amisulpride -, serotonin -] mean 5.3% ± 3.2% SEM, [atropine -, amisulpride -, serotonin +] mean 94.5% ± 0.9% SEM, [atropine +, amisulpride -, serotonin -] mean 1.7% ± 1.7% SEM, [atropine +, amisulpride -, serotonin +] mean 74.8% ± 14.8% SEM, [atropine -, amisulpride +, serotonin -] mean 2.9% ± 1.5% SEM, [atropine -, amisulpride +, serotonin +] mean 2.9% ± 1.5% SEM, anus: [atropine -, amisulpride -, serotonin -] mean 6.8% ± 1.8% SEM, [atropine -, amisulpride -, serotonin +] mean 5.9% ± 2.6% SEM, [atropine +, amisulpride -, serotonin -] mean 51.5% ± 11.6% SEM, [atropine +, amisulpride -, serotonin +] mean 38.6% ± 6.1% SEM, [atropine -, amisulpride +, serotonin -] mean 51.9% ± 14.6% SEM, [atropine -, amisulpride +, serotonin +] mean 74.1% ± 8.2% SEM). d Diagram illustrating the signaling pathways from light perception to pyloric and anal opening, mediated by Opsin proteins and neurotransmitters. The interplay between serotonin and dopamine suggests cross-talk between the pathways. e Schematic representation of the putative signaling pathway from photoreceptor cells to the pylorus and anus. Although the exact pathway from Opsin2 to the anus has not yet been identified, it has been reported that the signal from Opsin3.2 photoreception is mediated by serotonergic and nitric oxide (NO) neurons14. Opsin2-expressing cells appear to directly connect to cholinergic neurons in the arm region and may also have the potential to secrete unidentified diffusible molecules that mediate the photoreception signal to anal neurons. Statistical significance is indicated as p < 0.01, *p < 0.001; n.s. = not significant, Welch’s t test (two-sided). Error bars shown in all graphs indicate SEM. Source data are provided as a Source Data file.
	Fig. 6. Wavelength dependency in the regulation of pyloric and anal openings.a Differential regulation of pyloric and anal openings by blue and longer wavelengths, respectively. The inset in the upper right corner details the wavelengths of LED lights utilized in the experiments, and the photon flux density of all LED lights and artificial sunlight was adjusted to 500 µmol m−2 s−1 (N = 5 batches [each consisting of a different male and female pair], n [control] = 37, 81, 39, 41, 13 larvae, n [artificial sunlight] = 34, 33, 18, 43, 21 larvae, n [blue] = 23, 45, 30, 51, 33 larvae, n [green] = 30, 26, 23, 41, 19 larvae, n [yellow] = 28, 60, 23, 41, 24 larvae, n [red] = 37, 26, 24, 27, 39 larvae; pylorus: [control] mean 1.5% ± 1% SEM, [artificial sunlight] mean 12.2% ± 1.1% SEM, [blue] mean 22.6% ± 4.1% SEM, [green] mean 6.7% ± 1.7% SEM, [yellow] mean 4.1% ± 0.3% SEM, [red] mean 4.1% ± 0.9% SEM, anus: [control] mean 6.9% ± 1.8% SEM, [artificial sunlight] mean 27% ± 4% SEM, [blue] mean 26.2% ± 3.8% SEM, [green] mean 23.3% ± 4.9% SEM, [yellow] mean 20.2% ± 3.3% SEM, [red] mean 8.7% ± 3.2% SEM). b Absorption spectra of purified Opsin3.2 measured before (black curve) or after (red curve) blue light (460 nm) irradiation. (inset) Spectral changes of Opsin3.2 caused by blue light (460 nm) irradiation (red curve) and subsequent red light (>600 nm) irradiation (blue curve). These mirror-image changes were obtained using DDM-solubilized cell membranes expressing Opsin3.2 after the addition of 11-cis retinal. c Absorption spectra of purified Opsin2 measured before irradiation (black curve) or after yellow light (>500 nm) (red curve) and subsequent violet light (420 nm) (blue curve) irradiation. (inset) Spectral changes of purified Opsin2 caused by yellow light irradiation (red curve) and subsequent violet light irradiation (blue curve). The expression of Opsin2 in cultured cells was also confirmed by Western blot using Rho1D4 antibody (Supplementary Fig. 8). d Illustration of the distinct pathways activated by blue light leading to pyloric opening, and by longer wavelengths leading to anal opening, demonstrating wavelength-specific regulatory mechanisms. We used one-way ANOVA followed by Tukey’s post hoc test in a. Statistical significance denoted as p < 0.05, *p < 0.01; n.s. = not significant. Error bars shown in all graphs indicate SEM. Source data are provided as a Source Data file.
	Fig. 7. Evolution of through-gut.The acquisition of a through-gut in the stem lineage of bilaterians was accompanied by the evolution of a sphincter regulatory system, facilitating the unidirectional flow of digestive contents. This development contrasts with cnidarians, which utilize a single orifice for both ingestion and expulsion, and Xenacoelomorphs, characterized by a blind gut. Additionally, it is intriguing to consider that this sphincter regulatory mechanism might have been influenced by external environmental stimuli, such as light.

	Fig. 1. Light-induced anus opening in sea urchin larvae.a Lateral view of a sea urchin larva with the digestive tract illuminated, showing key anatomical features: the cardiac sphincter (, asterisk), pyloric sphincter (yellow), and anal sphincter (green). The inset on the bottom left depicts the anus in a closed state (arrowhead), while the bottom right shows the anus open and the intestine (i) contracted, 30 seconds post-photoirradiation. Annotations: M, mouth; e, esophagus; s, stomach; i, intestine. b Anus opening rates post-photoirradiation, assessed using anti-Troponin I (TnI) antibody staining (refer to Methods). The graph presents basic photoirradiation conditions alongside opening rates, with pyloric (blue) and anal (black) openings (N = 4 batches [each consisting of a different male and female pair], n [0 min] = 27, 32, 36, 46 larvae, n [1 min] = 18, 23, 43, 33 larvae, n [2 min] = 18, 37, 30, 45 larvae, n [3 min] = 14, 24, 42, 49 larvae, n [4 min] = 15, 11, 34, 39 larvae, n [5 min] = 21, 20, 40, 42 larvae, n [6 min] = 21, 25, 45, 36 larvae, n [7 min] = 24, 26, 27, 30 larvae, n [8 min] = 16, 27, 44, 40 larvae, n [9 min] = 20, 24, 52, 45 larvae, n [10 min] = 19, 25, 41, 44 larvae; pylorus: [0 min] mean 1.6% ± 1.6% SEM, [1 min] mean 11.1% ± 4.7% SEM, [2 min] mean 18.9% ± 1.2% SEM, [3 min] mean 10.8% ± 2.1% SEM, [4 min] mean 5% ± 1.9% SEM, [5 min] mean 4.8% ± 1.9% SEM, [6 min] mean 5.4% ± 2.3% SEM, [7 min] mean 3.8% ± 0.2% SEM, [8 min] mean 0.6% ± 0.6% SEM, [9 min] mean 0.6% ± 0.6% SEM, [10 min] mean 0.6% ± 0.6% SEM; anus: [0 min] mean 8.2% ± 2.2% SEM, [1 min] mean 31% ± 11% SEM, [2 min] mean 49.7% ± 8.9% SEM, [3 min] mean 46.3% ± 5.4% SEM, [4 min] mean 30.6% ± 4.4% SEM, [5 min] mean 34.8% ± 6.9% SEM, [6 min] mean 18.4% ± 3.7% SEM, [7 min] mean 16.3% ± 3.5% SEM, [8 min] mean 27.7% ± 11.2% SEM, [9 min] mean 14.5% ± 4.8% SEM, [10 min] mean 24.8% ± 4.1% SEM). c. Schematic of the experimental setup brain and pre-oral arm ablation with a needle. Color coding: orange, brain; blue, Opsin3.2-expressing cells; magenta, Opsin2-expressing cells; yellow, pylorus; green, anus. The graph illustrates the opening rates of the pylorus and anus 2 minutes after photoirradiation (N = 5 batches, n [control] = 95, 38, 54, 49, 37 larvae, n [brain & pre-oral arms removed] = 54, 22, 27, 28, 25 larvae; pylorus: [control] mean 17.2% ± 4.1% SEM, [brain & pre-oral arms removed] mean 3.5% ± 2.1% SEM; anus: [control] mean 40.7% ± 2.5% SEM, [brain & pre-oral arms removed] mean 58.8% ± 5.7% SEM). d, e Graphs depicting the opening rates of the pylorus and anus under various conditions: d serotonin treatment (10 µM) without photoirradiation (N = 4 batches, n [serotonin -] = 53, 42, 49, 53 larvae, n [serotonin +] = 35, 48, 45, 58 larvae; pylorus: [serotonin -] mean 4.3% ± 2% SEM, [serotonin +] mean 82.5% ± 7.7% SEM; anus: [serotonin -] mean 3.7% ± 1.2% SEM, [serotonin +] mean 5.5% ± 1.7% SEM); e serotonin treatment (10 µM) with photoirradiation (N = 3 batches, n [serotonin -] = 147, 71, 94 larvae, n [serotonin +] = 53, 31, 34 larvae; pylorus: [serotonin -] mean 20.4% ± 3.8% SEM, [serotonin +] mean 87.6% ± 11.5% SEM; anus: [serotonin -] mean 36.5% ± 5.8% SEM, [serotonin +] mean 2.6% ± 1.7% SEM). Statistical significance denoted as p < 0.05, **p < 0.01; n.s. = not significant, Welch’s t test (two-sided). Error bars shown in all graphs indicate SEM. Scale bar in a = 50 µm and in b =20 µm. Source data are provided as a Source Data file.
	Fig. 2. Light-induced anus opening is mediated by Opsin2.a Opsin2 cells are missing in Opsin2 morphants. Anus opening or closing is identified with anti-TroponinI (TnI) antibody (arrowhead). b–d Graphs depicting the opening rates of the pylorus and anus under various conditions: b in control versus Opsin2 morphants post-photoirradiation (N = 4 batches [each consisting of a different male and female pair], n [control] = 38, 77, 53, 19 larvae, n [Opsin2 MO-1] = 16, 10, 10, 20 larvae; pylorus: [control] mean 20.3% ± 5.4% SEM, [Opsin2 MO-1] mean 28.8% ± 4.3% SEM; anus: [control] mean 23.5% ± 4.5% SEM, [Opsin2 MO-1] mean 5% ± 5% SEM); c with or without atropine treatment (100 µM) (N = 3 batches, n [atropine -] = 57, 118, 61 larvae, n [atropine +] = 18, 42, 31 larvae; pylorus: [atropine -] mean 5% ± 2.5% SEM, [atropine +] mean 2.9% ± 1.6% SEM; anus: [atropine -] mean 6.2% ± 1.6% SEM, [atropine +] mean 34.1% ± 0.7% SEM); d with or without acetylcholine treatment (1 µM) with photoirradiation (N = 7 batches, n [acetylcholine -] = 38, 104, 70, 115, 114, 92, 147 larvae, n [acetylcholine +] = 46, 23, 40, 36, 33, 25, 62 larvae; pylorus: [acetylcholine -] mean 19.7% ± 4.3% SEM, [acetylcholine +] mean 29.2% ± 4.6% SEM; anus: [acetylcholine -] mean 39.5% ± 1.6% SEM, [acetylcholine +] mean 8% ± 3.6% SEM). e Illustration of the signaling pathways from light perception to pyloric and anal opening, mediated by Opsin proteins and neurotransmitters. Statistical significance denoted as p < 0.05, **p < 0.001; n.s. = not significant, Welch’s t test (two-sided). Error bars shown in all graphs indicate SEM. Scale bar in a =20 µm. Source data are provided as a Source Data file.
	Fig. 3. ChAT-positive anal neurons in sea urchin larvae.a The graph illustrates the number of anal neurons over time, showing the presence of approximately two neurons at the anus starting three days post-fertilization. (N = 3 batches [each consisting of a different male and female pair], n [2-day] = 4, 4, 3 larvae, n [3-day] = 6, 5, 6 larvae, n [4-day] = 8, 13, 3 larvae, n [5-day] = 4, 7, 3 larvae, n [6-day] = 8, 10, 10 larvae, n [7-day] = 7, 10, 8 larvae; [2 day] mean 0% ± 0% SEM, [3 day] mean 1.4% ± 0.1% SEM, [4 day] mean 1.9% ± 0.1% SEM, [5 day] mean 2% ± 0% SEM, [6 day] mean 1.9% ± 0.2% SEM, [7 day] mean 2.1% ± 0.1% SEM). b Immunostaining images of anal and intestine (yellow dotted-line) region with anti-SynB antibody (neurons) and anti-TroponinI (TnI) antibody (muscles). A rectangle indicated the magnified region shown in the right. The asterisks in the magnified view highlight the neurons. The schematic diagram presents a lateral view of the stomach and intestine. Magenta cells and green areas indicate neurons and sphincters, respectively, at anus. c–f Distribution of cholinergic neurons within the sea urchin larval intestine. d ChAT-neurons are not present around the pylorus. e Asterisks highlight neurons surrounding the anus. f Magenta cells in the schematic image depict ChAT-positive anal neurons. Scale bar in b =10 µm; c =20 µm. Source data are provided as a Source Data file.
	Fig. 4. TH-positive pyloric and anal neurons in sea urchin larvae.a The expression pattern of tyrosine hydroxylase (TH) mRNA in 60-hour larvae. TH is a rate-limiting enzyme for dopamine biosynthesis, with dopamine neurons present at the ciliary band, pylorus (arrowheads), and anus (asterisks). Magenta cells in the schematic image represent dopamine neurons at the pylorus and anus. b Arrows indicate stomach-side enteric neurons (sEN); arrowheads point to intestine-side enteric neurons (iEN); asterisks highlight neurons surrounding the anus. c Anal neurons (asterisks) co-express ChAT and TH. d Schematic representation of the sea urchin larval stomach and intestine, emphasizing neurons near the pylorus (yellow) and anus (green). Neuron types are color-coded: sEN (orange) as TH-/ChAT-, iEN (blue) as TH + /ChAT-, and perianal neurons (magenta) as TH + /ChAT + . Scale bar in a, b, and c =20 µm. Source data for micrograph are provided as a Source Data file.
	Fig. 5. Interactions between pyloric and anal opening pathways in sea urchin larvae.a Opening rates of the pylorus and anus in larvae treated with or without dopamine (0.1 µM) with photoirradiation (N = 4 batches [each consisting of a different male and female pair], n [dopamine -] = 114, 72, 71, 94 larvae, n [dopamine +] = 40, 29, 33, 53 larvae; pylorus: [dopamine -] mean 30.5% ± 4% SEM, [dopamine +] mean 36% ± 4.8% SEM; anus: [dopamine -] mean 35.8% ± 4% SEM, [dopamine +] mean 7.8% ± 0.5% SEM). b Opening rates of the pylorus and anus in larvae treated with or without the dopamine inhibitor amisulpride (50 µM) (N = 5 batches, n [amisulpride -] = 38, 67, 40, 47, 46 larvae, n [amisulpride +] = 20, 55, 48, 41 36 larvae; pylorus: [amisulpride -] mean 6.2% ± 1.6% SEM, [amisulpride +] mean 2.6% ± 2.2% SEM; anus: [amisulpride -] mean 5.2% ± 1.2% SEM, [amisulpride +] mean 18.4% ± 2.9% SEM). c Opening rates of the pylorus and anus in larvae exposed to a combination of inhibitors (100 µM atropine or 50 µM amisulpride) and serotonin (10 µM; applied 5 min after inhibitor treatment), illustrating the modulatory effects on opening behavior (N = 3 batches, n [atropine -, amisulpride -, serotonin -] = 27, 21, 30 larvae, n [atropine -, amisulpride -, serotonin +] = 27, 31, 31 larvae, n [atropine +, amisulpride -, serotonin -] = 20, 20, 23 larvae, n [atropine +, amisulpride -, serotonin +] = 19, 31, 24 larvae, n [atropine -, amisulpride +, serotonin -] = 21, 10, 26 larvae, n [atropine -, amisulpride +, serotonin +] = 23, 30, 17 larvae; pylorus: [atropine -, amisulpride -, serotonin -] mean 5.3% ± 3.2% SEM, [atropine -, amisulpride -, serotonin +] mean 94.5% ± 0.9% SEM, [atropine +, amisulpride -, serotonin -] mean 1.7% ± 1.7% SEM, [atropine +, amisulpride -, serotonin +] mean 74.8% ± 14.8% SEM, [atropine -, amisulpride +, serotonin -] mean 2.9% ± 1.5% SEM, [atropine -, amisulpride +, serotonin +] mean 2.9% ± 1.5% SEM, anus: [atropine -, amisulpride -, serotonin -] mean 6.8% ± 1.8% SEM, [atropine -, amisulpride -, serotonin +] mean 5.9% ± 2.6% SEM, [atropine +, amisulpride -, serotonin -] mean 51.5% ± 11.6% SEM, [atropine +, amisulpride -, serotonin +] mean 38.6% ± 6.1% SEM, [atropine -, amisulpride +, serotonin -] mean 51.9% ± 14.6% SEM, [atropine -, amisulpride +, serotonin +] mean 74.1% ± 8.2% SEM). d Diagram illustrating the signaling pathways from light perception to pyloric and anal opening, mediated by Opsin proteins and neurotransmitters. The interplay between serotonin and dopamine suggests cross-talk between the pathways. e Schematic representation of the putative signaling pathway from photoreceptor cells to the pylorus and anus. Although the exact pathway from Opsin2 to the anus has not yet been identified, it has been reported that the signal from Opsin3.2 photoreception is mediated by serotonergic and nitric oxide (NO) neurons14. Opsin2-expressing cells appear to directly connect to cholinergic neurons in the arm region and may also have the potential to secrete unidentified diffusible molecules that mediate the photoreception signal to anal neurons. Statistical significance is indicated as p < 0.01, *p < 0.001; n.s. = not significant, Welch’s t test (two-sided). Error bars shown in all graphs indicate SEM. Source data are provided as a Source Data file.
	Fig. 6. Wavelength dependency in the regulation of pyloric and anal openings.a Differential regulation of pyloric and anal openings by blue and longer wavelengths, respectively. The inset in the upper right corner details the wavelengths of LED lights utilized in the experiments, and the photon flux density of all LED lights and artificial sunlight was adjusted to 500 µmol m−2 s−1 (N = 5 batches [each consisting of a different male and female pair], n [control] = 37, 81, 39, 41, 13 larvae, n [artificial sunlight] = 34, 33, 18, 43, 21 larvae, n [blue] = 23, 45, 30, 51, 33 larvae, n [green] = 30, 26, 23, 41, 19 larvae, n [yellow] = 28, 60, 23, 41, 24 larvae, n [red] = 37, 26, 24, 27, 39 larvae; pylorus: [control] mean 1.5% ± 1% SEM, [artificial sunlight] mean 12.2% ± 1.1% SEM, [blue] mean 22.6% ± 4.1% SEM, [green] mean 6.7% ± 1.7% SEM, [yellow] mean 4.1% ± 0.3% SEM, [red] mean 4.1% ± 0.9% SEM, anus: [control] mean 6.9% ± 1.8% SEM, [artificial sunlight] mean 27% ± 4% SEM, [blue] mean 26.2% ± 3.8% SEM, [green] mean 23.3% ± 4.9% SEM, [yellow] mean 20.2% ± 3.3% SEM, [red] mean 8.7% ± 3.2% SEM). b Absorption spectra of purified Opsin3.2 measured before (black curve) or after (red curve) blue light (460 nm) irradiation. (inset) Spectral changes of Opsin3.2 caused by blue light (460 nm) irradiation (red curve) and subsequent red light (>600 nm) irradiation (blue curve). These mirror-image changes were obtained using DDM-solubilized cell membranes expressing Opsin3.2 after the addition of 11-cis retinal. c Absorption spectra of purified Opsin2 measured before irradiation (black curve) or after yellow light (>500 nm) (red curve) and subsequent violet light (420 nm) (blue curve) irradiation. (inset) Spectral changes of purified Opsin2 caused by yellow light irradiation (red curve) and subsequent violet light irradiation (blue curve). The expression of Opsin2 in cultured cells was also confirmed by Western blot using Rho1D4 antibody (Supplementary Fig. 8). d Illustration of the distinct pathways activated by blue light leading to pyloric opening, and by longer wavelengths leading to anal opening, demonstrating wavelength-specific regulatory mechanisms. We used one-way ANOVA followed by Tukey’s post hoc test in a. Statistical significance denoted as p < 0.05, *p < 0.01; n.s. = not significant. Error bars shown in all graphs indicate SEM. Source data are provided as a Source Data file.
	Fig. 7. Evolution of through-gut.The acquisition of a through-gut in the stem lineage of bilaterians was accompanied by the evolution of a sphincter regulatory system, facilitating the unidirectional flow of digestive contents. This development contrasts with cnidarians, which utilize a single orifice for both ingestion and expulsion, and Xenacoelomorphs, characterized by a blind gut. Additionally, it is intriguing to consider that this sphincter regulatory mechanism might have been influenced by external environmental stimuli, such as light.