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. 2022 Aug 8;6(10):1438–1448. doi: 10.1038/s41559-022-01835-7

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Extended Data Fig. 4 — Neuropeptide immunofluorescent staining of adult V. multiformis. a, Results of immunofluorescent are summarized as in the schematic diagram of V. multiformis. The left half shows the aboral view and the right half shows the oral. Abbreviations: ao, apical organ; tc, tentacle; tb, tentacle bulb; mo, mouth. The distributions of each peptide-expressing cell type are indicated in different colors. b–d, subepithelial neural network on the aboral surface (b, magnified view in c) and the oral surface (d) are visualised by the VWYa staining. e–e”, VWYa⁺ neurons (green) develop polygonal neural mesh in the dorsal subepithelial layer where tubulin staining (magenta) is partially overlapped. f, Distribution of VWYa positive neural net-like structures (green) and phalloidin positive protrusions (magenta) on the aboral surface. g–g”, Some of these protrusions (magenta) are extended from the VWYa⁺ cell bodies (green). h, This neural network-like pattern was observed on the surface of the tentacle bulb. i, VWYa⁺ neural network-like pattern (green) was also observed in the tentacle. j, Cell bodies of VWYa⁺ neurons (green) bearing F-actin (phalloidin)-positive protrusion (magenta) are located on the surface of the tentacle. k,m, NPWa⁺ cells distributed sparsely on the aboral (k) and oral (m) epithelial surface. The distribution of NPWa was not observed on the neurite-like structure, but in the short processes in some of the positive cells, especially on the aboral side (arrowheads in k). NPWa⁺ cells (green in k) are not associated with phalloidin positive protrusion of F-actin (magenta in k). l–l”, Double immunostaining of VWYa and NPWa. The distribution of VWYa⁺ (green) and NPWa⁺ (magenta) were not overlapped but cells positive for each peptide were closely associated (magnified in inset). n,o, Distribution of FGLa⁺ cells was observed on the body margin. FGLa⁺ cells contained large spherical organelles in their cell bodies (o). p,q, Distribution of WTGa. WTGa⁺ cells were small round shapes and sparsely distributed on the oral surface of the animal. The frequency of these cells was higher in the outer region. Schematic diagrams inserted in the panels indicate the position of the following images in the animal. Scale bars are 20 μm (c, e–o and q) and 50 μm (b, d and p). Pseudo-colours in b–q were applied by ImageJ software.