Yaguchi J et al. (2025), Non-visual photoreceptive brain specification i...

ECB-ART-54468

Nat Commun 2025 Nov 19;161:10054. doi: 10.1038/s41467-025-65628-9.

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Non-visual photoreceptive brain specification in sea urchin larvae.

Yaguchi J, Tsuyuzaki K, Sawada I, Horiuchi A, Sakamoto N, Yamamoto T, Yamashita T, Yaguchi S.

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Centralized nervous systems enable animals to detect environmental cues and coordinate behavior, but their evolutionary origins in deuterostomes remain unclear. Among deuterostomes, echinoderms-such as sea urchins-have long been thought to lack brain-like structures, especially in larval stages. Although recent gene expression and neural activity studies suggest brain-like properties in sea urchin larvae, direct links to behavior are still emerging. Here, we identify a light-sensitive cluster of neurons in the posterior neuroectoderm of sea urchin larvae. These neurons express UV-sensitive Opsin5 and regulatory genes such as rx, otx, six3, and lhx6, which are conserved in the vertebrate diencephalon. We mapped this domain using single-cell RNA sequencing and in situ hybridization. Knockdown of Opn5L impaired light-dependent swimming, indicating an active role in photoreception. While further work is needed to fully establish circuit-to-behavior relationships, our findings add to growing evidence that sea urchin larvae possess a non-visual photoreceptive neural center with molecular features shared by vertebrate brain regions. This suggests that such domains originated in the deuterostome ancestor and contributed to the early evolution of brain function.

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	Fig. 1. Photoreceptor-positive and -negative serotonergic neurons are present in the brain of sea urchin larvae.a A simplified phylogenetic tree indicating that non-visual photoreceptive brain structures are present in chordates but remain unclear in echinoderms and hemichordates. b Schematic overview of the experimental design for scRNA-seq with DAPT treatment from 24 (gastrula) to 96 hours (pluteus). In the UMAP plot, magenta represents control cells, while green represents DAPT-treated cells. c–e A UMAP visualization of scRNA-seq data from control samples at 24 to 96 hours, with different color coding. As explained in Supplementary Fig. 1, all UMAPs presented in this paper represent control-only data derived from the control-DAPT integrated dataset, with DAPT-subtracted. The magnified region within the rectangle in (c) is shown in (c’), where neurons are highlighted in brown. c” Only tph-expressing cells are highlighted in blue. tph-expressing cells are clustered into two (d), while opn5L is expressed in only one of them (e). Light blue dotted lines, anterior serotonergic neurons; orange lines, dorsal serotonergic neurons. f. Expression patterns of tph and opn5L in 36-hour larvae, viewed dorsally. Cells co-expressing tph and opn5L are located in the dorsal region (orange-lined rectangle), distinct from the anterior region, where tph is expressed alone (light blue dotted-line rectangles). The confocal images f’ and f” correspond to the gene expression regions shown in the UMAP (d, e). The box region in f is magnified in f’ and f”. g Expression patterns of opn5L and foxQ2 mRNA viewed laterally in 36-hour (g, g’). Bar = 20 µm. h Expression patterns of opn5L and synaptotagminB (synB) viewed laterally in 48-hour larvae (h, h’). The magnified views in g’ and h’ show that opn5L-expressing cells are dorsally positioned, separate from the foxQ2-expressing region as well as synB cells (asterisk), which represent anterior serotonergic neurons at this stage. Arrows indicate opn5L-expressing cells. V, ventral; D, dorsal. Expression patterns of SynaptotagminB (SynB) and serotonin in 36-hour to 72-hour larvae, viewed dorsally (i–n) and laterally (o–p’). Anterior serotonergic neurons (asterisks) in the larval apical region first appear around 36 hours (i). Dorsal serotonergic neurons (arrows) emerge from 42-hour larvae (j) and gradually move closer to the anterior serotonergic neurons by 54 hours (k). The percentage of dorsal serotonergic neurons detected was 1.5% in 36-hour larvae (n = 136), 43.7% in 42-hour larvae (n = 126), and 87.3% in 48-hour larvae (n = 63). By 72 hours, dorsal serotonergic neurons became difficult to detect without sectioning (n). The inset in n shows a dorsal section of the central rectangle area in n. Lateral views clearly distinguish dorsal serotonergic neurons (arrows) from anterior serotonergic neurons (asterisks) in both 48-hour (o, o’) and 72-hour (p, p’) larvae. q Schematic representation of anterior serotonergic neurons (green) and dorsal serotonergic neurons (yellow), shown in dorsal and lateral views.
	Fig. 2. Gene expression patterns and function of Z167 in dorsal serotonergic neurons, in which opn5L is expressed.a Co-expression of opn5L and z167 at 48-hour pluteus larva. Bar = 20 µm. a’, a” Magnified image of a square in (a). b Expression pattern of z167 in 30-hour late gastrula (LG). Detection of z167 began around 30 hours, with 43.3% of larvae (n = 203) showing z167 signals. c Co-expression pattern of z167 and foxQ2 in 36-hour prism larvae. d Co-expression pattern of ebf3 and foxQ2 in 36-hour larvae. Co-expression pattern of z167 and ebf3 in 48-hour pluteus larvae under control conditions (e) and with DAPT treatment (f). A, anterior; P, posterior. g UMAP representation of z167- and ebf3-expressing cells. h–j. Co-expression pattern of z167 and tph in 36- to 54-hour larvae. Expression of tph in dorsal serotonergic neurons was transient, and the tph signal was nearly undetectable in 54-hour larvae (j, j’, arrows). The number of tph-positive cells in the dorsal region was 1.53 ± 0.22 (N = 3, n = 53, 27, 7) in 48-hour larvae and 0.58 ± 0.24 (N = 3, n = 10, 14, 15) in 54-hour larvae. In contrast, z167 expression persisted beyond 54 hours, suggesting that the z167 probe serves as a reliable marker for dorsal serotonergic neurons. The number of z167-positive cells was 3.1 (n = 39), with 100% detection in 54-hour larvae. Arrows indicate dorsal serotonergic neurons, and asterisks indicate anterior serotonergic neurons. k, l Expression patterns of serotonin and Synaptotagmin B (SynB) in control larvae (o) and z167 morphants (l). In z167 morphants, SynB (neuronal marker) was detected in the region where dorsal serotonergic neurons are typically found, whereas serotonin signals were absent (l–l”). The boxed regions in k and l are magnified in k’, k” and l’, l”, respectively. Arrows indicate dorsal serotonergic neurons.
	Fig. 3. Co-expression genes in dorsal serotonergic neurons with z167/opn5L.a–h Co-expression patterns of sna, hmx, six3, awh (lhx6), otx, cry1, and rx with z167, which exhibited relatively high expression levels in dorsal serotonergic neurons based on single-cell analysis in 48-hour pluteus larvae treated with DMSO (control). All factors were detected in the z167, i.e., opn5L-expressing cells. a-a” Co-expression pattern of sna with z167 in 48-hour larvae. Bar = 20 µm. b-b” Co-expression pattern of hmx with z167 in 48-hour larvae. c-c”. Co-expression pattern of six3 with z167 in 48-hour larvae. d-d” Co-expression pattern of limc1 with z167 in 48-hour larvae. e-e” Co-expression pattern of awh with z167 in 48-hour larvae. f-f”. Co-expression pattern of otx with z167 in 48-hour larvae. g-g” Co-expression pattern of cry1 with z167 in 48-hour larvae. h-h” Co-expression pattern of rx and z167 in 48-hour larvae. a’-h” Magnified image of boxed area in (a–h), respectively. All experiments were independently confirmed in more than 3 batches.
	Fig. 4. Potential Snail-mediated migration of dorsal serotonergic neurons toward the anterior region.a, b UMAP representation of snail (sna) expression. sna was expressed in the skeleton, pigment cells, coelomic pouch, and the dorsal serotonergic neurons. c Co-expression pattern of sna and gcm (a marker for secondary mesenchyme cells) in 33-hour larvae. These images clearly demonstrate that sna is expressed in the skeleton (red arrows), coelomic pouch (ocher arrow), and neurons (white arrow). V, ventral; D, dorsal. Bar = 20 µm. The boxed area in (c) is magnified in c’- c”’. d Co-expression pattern of sna and z167 in 33-hour larvae, viewed dorsally. sna expression in the dorsal ectoderm completely overlapped with the z167 signal (white arrow). e, f Quantification of the spatial relationship between dorsal and anterior serotonergic neurons. In the co-expression pattern of z167 and tph, z167-positive cells were identified as dorsal serotonergic neurons, whereas tph-only cells were classified as anterior serotonergic neurons (e). The number of cells between these two neuronal populations was manually counted in confocal laser microscopy sections. The graph (f) shows that from 48-hour larvae, dorsal and anterior serotonergic neurons began to move closer together. The proportion of dorsal serotonergic neurons in direct contact with anterior serotonergic neurons was 0% at 36 hours (n = 56), 1.5% at 42 hours (n = 65), 11.1% at 48 hours (n = 81), and 51.9% at 54 hours (n = 52). The mean number of cells located between dorsal and anterior serotonergic neurons was: 36-hour larvae: 2.54 ± 0.15 SEM, 42-hour larvae: 2.38 ± 0.13 SEM, 48-hour larvae: 1.94 ± 0.13 SEM, and 54-hour larvae: 0.73 ± 0.13 SEM. A one-way ANOVA followed by Tukey’s post hoc test was used for statistical analysis. Error bars indicate S.E. ** [36 h vs 48 h], p = 0.009274, [36 h vs 54 h], p = 0.0010053; n.s. = not significant [36 h vs 42 h], p = 0.8565991. g, h Dorsal serotonergic neurons were detected on the inner surface of the ectodermal layer. In lateral views, z167-expressing cells were occasionally observed on the inner side of the ectodermal layer, as indicated by the arrow in (g’). The boxed region in g is magnified in g’. Similarly, serotonin-expressing cells were also found attached to the inner surface of the ectodermal layer (arrows in h, h’). These cells lacked Epith2 signal, an epithelial cell surface molecular marker. Asterisks indicate anterior serotonergic neurons.
	Fig. 5. Opn5L function and the potential evolutionary scenario for non-visual photoreceptive brain regional specification.a, b Expression patterns of serotonin and Synaptotagmin B (Syn B) in control and Opn5L-MO1-injected larvae. Bar = 20 µm. Opn5L-MO1-injected larvae developed into pluteus larvae similar to controls, with both anterior and dorsal serotonergic neurons observed normally (b, b’). The boxed region in (b) is magnified in (b’). Arrows indicate dorsal serotonergic neurons. c Differences in floating rates between control and Opn5L-MO1-injected larvae under dark and light conditions. Control and Opn5L morphants were cultured in light or dark conditions from 24 hours post-fertilization, and the number of actively swimming larvae (without sinking) was counted at the four-day larval stage. Control larvae continued to swim in the dark, but their swimming rates decreased significantly under light conditions. In contrast, Opn5L morphants maintained high swimming rates under both dark and light conditions. Photon flux density was set to 150–200 μmol m⁻² s⁻¹. The mean swimming rate in control larvae was 95.9% ± 2.1% SEM (n = 56, 33, 27, 49, 58) in the dark and 34.7% ± 7.5% SEM (n = 38, 41, 51, 32, 59) in the light. Opn5L-MO1-injected larvae showed a mean swimming rate of 96.5% ± 0.9% SEM (n = 22, 19, 22, 24, 60) in the dark and 85.7% ± 8.0% SEM (n = 48, 58, 38, 18, 61) in the light. **, p = 0.0007504; n.s., not significant, p = 0.2487. d Molecular property of H. pulcherrimus Opn5L protein. (upper panel) Spectral property of H. pulcherrimus Opn5L protein. Absorption spectra of Opn5L protein purified after the addition of 11-cis retinal were recorded in the dark (curve 1), after UV light (360 nm) irradiation (curve 2) and after subsequent yellow light (>500 nm) irradiation (curve 3). UV light irradiation shifted the spectrum from the UV region (~390 nm) to the visible region (~500 nm), and subsequent yellow light irradiation recovered the absorbance in the UV region. Combined with the results for S. purpuratus Opn5L protein (see Supplementary Fig. 7), sea urchin Opn5L is a UV-sensitive bistable opsin like vertebrate Opsin5. (inset) Spectral changes of H. pulcherrimus Opn5L protein caused by UV light irradiation (curve 1) and subsequent yellow light irradiation (curve 2). (lower panel) Opn5L-evoked elevation of intracellular Ca2+ level. The Ca2+ level change in H. pulcherrimus Opn5L-transfected (red curve) or mock-transfected (black curve) HEK293 cells was measured using an aequorin-based luminescent assay. Data are presented as the means of two independent experiments. A transient elevation of the luminescence by light irradiation from UV LED light (365 nm) was observed in Opn5L-expressing cells. This means that H. pulcherrimus Opn5L triggers the intracellular Ca2+ signaling in a light-dependent manner like vertebrate Opsin5. e Conserved gene expression patterns (see Supplementary Fig. 8) in the non-visual photoreceptive brain region between echinoderms and chordates. Gray text indicates genes that are not expressed. While the process and/or pattern of neuronal internalization differs between the two groups, this variation may be linked to differences in the complexity of brain development and evolution. Light blue, future telencephalon (-like); orange, future diencephalon (-like).