{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,7,30]],"date-time":"2025-07-30T15:48:28Z","timestamp":1753890508525,"version":"3.41.2"},"reference-count":37,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2021,7,26]],"date-time":"2021-07-26T00:00:00Z","timestamp":1627257600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001824","name":"Grantov\u00e1 Agentura \u010cesk\u00e9 Republiky","doi-asserted-by":"publisher","id":[{"id":"10.13039\/501100001824","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell Dev. Biol."],"abstract":"<jats:p>Cephalochordates (amphioxi or lancelets) are representatives of the most basally divergent group of the chordate phylum. Studies of amphioxus development and anatomy hence provide a key insight into vertebrate evolution. More widespread use of amphioxus in the evo\u2013devo field would be greatly facilitated by expanding the methodological toolbox available in this model system. For example, evo\u2013devo research on amphioxus requires deep understanding of animal anatomy. Although conventional confocal microscopy can visualize transparent amphioxus embryos and early larvae, the imaging of later developmental stages is problematic because of the size and opaqueness of the animal. Here, we show that light sheet microscopy combined with tissue clearing methods enables exploration of large amphioxus specimens while keeping the surface and the internal structures intact. We took advantage of the phenomenon of autofluorescence of amphioxus larva to highlight anatomical details. In order to investigate molecular markers at the single-cell level, we performed antibody-based immunodetection of melanopsin and acetylated-\u03b1-tubulin to label rhabdomeric photoreceptors and the neuronal scaffold. Our approach that combines light sheet microscopy with the clearing protocol, autofluorescence properties of amphioxus, and antibody immunodetection allows visualizing anatomical structures and even individual cells in the 3D space of the entire animal body.<\/jats:p>","DOI":"10.3389\/fcell.2021.702986","type":"journal-article","created":{"date-parts":[[2021,7,26]],"date-time":"2021-07-26T12:28:22Z","timestamp":1627302502000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":0,"title":["Optical Clearing and Light Sheet Microscopy Imaging of Amphioxus"],"prefix":"10.3389","volume":"9","author":[{"given":"Simona","family":"Machacova","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Helena","family":"Chmelova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna","family":"Vavrova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zbynek","family":"Kozmik","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iryna","family":"Kozmikova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1965","published-online":{"date-parts":[[2021,7,26]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"1679","DOI":"10.1038\/nprot.2015.111","article-title":"Efficient processing and analysis of large-scale light-sheet microscopy data.","volume":"10","author":"Amat","year":"2015","journal-title":"Nat. Protoc."},{"key":"B2","doi-asserted-by":"publisher","first-page":"255","DOI":"10.2307\/1543136","article-title":"A High-Affinity Hemoglobin Is Expressed in the Notochord of Amphioxus, Branchiostoma californiense.","volume":"195","author":"Bishop","year":"1998","journal-title":"Biol. Bull."},{"key":"B3","doi-asserted-by":"publisher","first-page":"77","DOI":"10.1186\/1471-2148-9-77","article-title":"Amphioxus encodes the largest known family of green fluorescent proteins, which have diversified into distinct functional classes.","volume":"9","author":"Bomati","year":"2009","journal-title":"BMC Evol. Biol."},{"key":"B4","doi-asserted-by":"publisher","first-page":"793","DOI":"10.1387\/ijdb.170259ik","article-title":"Novel polyclonal antibodies as a useful tool for expression studies in amphioxus embryos.","volume":"61","author":"Bozzo","year":"2017","journal-title":"Int. J. Dev. Biol."},{"key":"B5","doi-asserted-by":"publisher","first-page":"2211","DOI":"10.1002\/cne.23785","article-title":"Neuronal organization of the brain in the adult amphioxus (Branchiostoma lanceolatum): a study with acetylated tubulin immunohistochemistry.","volume":"523","author":"Castro","year":"2015","journal-title":"J. Comp. Neurol."},{"key":"B6","doi-asserted-by":"publisher","first-page":"69","DOI":"10.1002\/jmor.1050540103","article-title":"The embryology of amphioxus.","volume":"54","author":"Conklin","year":"1932","journal-title":"J. Morphol."},{"key":"B7","doi-asserted-by":"publisher","first-page":"95","DOI":"10.2307\/25066625","article-title":"Endogenous green fluorescent protein (GFP) in amphioxus.","volume":"213","author":"Deheyn","year":"2007","journal-title":"Biol. Bull."},{"key":"B8","doi-asserted-by":"publisher","first-page":"965","DOI":"10.1038\/nature04336","article-title":"Tunicates and not cephalochordates are the closest living relatives of vertebrates.","volume":"439","author":"Delsuc","year":"2006","journal-title":"Nature"},{"key":"B9","doi-asserted-by":"publisher","first-page":"319","DOI":"10.1002\/cne.10803","article-title":"Cytoarchitecture, topography, and descending supraspinal projections in the anterior central nervous system of Branchiostoma lanceolatum.","volume":"466","author":"Ekhart","year":"2003","journal-title":"J. Comp. Neurol."},{"key":"B10","doi-asserted-by":"publisher","first-page":"11088","DOI":"10.1038\/ncomms11088","article-title":"Imaging multicellular specimens with real-time optimized tiling light-sheet selective plane illumination microscopy.","volume":"7","author":"Fu","year":"2016","journal-title":"Nat. Commun."},{"journal-title":"Studien \u00fcber Entwicklung des Amphioxus.","year":"1881","author":"Hatschek","key":"B11"},{"key":"B12","doi-asserted-by":"publisher","first-page":"1963","DOI":"10.1242\/dev.022426","article-title":"Selective plane illumination microscopy techniques in developmental biology.","volume":"136","author":"Huisken","year":"2009","journal-title":"Development"},{"key":"B13","doi-asserted-by":"publisher","first-page":"394","DOI":"10.1002\/cne.1039","article-title":"Establishment of left-right asymmetric innervation in the lancelet oral region.","volume":"435","author":"Kaji","year":"2001","journal-title":"J. Comp. Neurol."},{"key":"B14","doi-asserted-by":"publisher","first-page":"2","DOI":"10.1186\/s40851-016-0038-3","article-title":"Amphioxus mouth after dorso-ventral inversion.","volume":"2","author":"Kaji","year":"2016","journal-title":"Zool. Lett."},{"key":"B15","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1086\/BBLv217n2p151","article-title":"Dynamic modification of oral innervation during metamorphosis in branchiostoma belcheri, the oriental lancelet.","volume":"217","author":"Kaji","year":"2009","journal-title":"Biol. Bull."},{"key":"B16","doi-asserted-by":"publisher","first-page":"624","DOI":"10.1016\/j.conb.2009.03.008","article-title":"Quantitative in vivo imaging of entire embryos with Digital Scanned Laser Light Sheet Fluorescence Microscopy.","volume":"18","author":"Keller","year":"2008","journal-title":"Curr. Opin. Neurobiol."},{"key":"B17","doi-asserted-by":"publisher","first-page":"1065","DOI":"10.1016\/j.cub.2005.04.063","article-title":"Cephalochordate melanopsin: evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells.","volume":"15","author":"Koyanagi","year":"2005","journal-title":"Curr. Biol."},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1016\/bs.mcb.2016.01.001","author":"Kromm","year":"2016","journal-title":"An Eye On Light-Sheet Microscopy."},{"key":"B19","first-page":"903","article-title":"Cell morphology in amphioxus nerve cord may reflect the time course of cell differentiation.","volume":"44","author":"Lacalli","year":"2000","journal-title":"Int. J. Dev. Biol."},{"key":"B20","doi-asserted-by":"publisher","first-page":"212","DOI":"10.1002\/jmor.10133","article-title":"Ventral neurons in the anterior nerve cord of amphioxus larvae. II. Further data on the pacemaker circuit.","volume":"257","author":"Lacalli","year":"2003","journal-title":"J. Morphol."},{"key":"B21","doi-asserted-by":"publisher","first-page":"1461","DOI":"10.1098\/rspb.1999.0801","article-title":"The oral nerve plexus in amphioxus larvae: function, cell types and phylogenetic significance.","volume":"266","author":"Lacalli","year":"1999","journal-title":"Proc. R. Soc. B Biol. Sci."},{"key":"B22","doi-asserted-by":"publisher","first-page":"165","DOI":"10.1098\/rstb.1994.0059","article-title":"Landmarks in the anterior central nervous system of amphioxus larvae.","volume":"344","author":"Lacalli","year":"1994","journal-title":"Philos. Trans. R. Soc. B Biol. Sci."},{"key":"B23","doi-asserted-by":"publisher","first-page":"16898","DOI":"10.1038\/srep16898","article-title":"High-resolution in-depth imaging of optically cleared thick samples using an adaptive SPIM.","volume":"5","author":"Masson","year":"2015","journal-title":"Sci. Rep."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1901891","DOI":"10.1002\/advs.201901891","article-title":"Fast, 3D Isotropic Imaging of Whole Mouse Brain Using Multiangle-Resolved Subvoxel SPIM.","volume":"7","author":"Nie","year":"2020","journal-title":"Adv. Sci."},{"key":"B25","doi-asserted-by":"publisher","first-page":"665","DOI":"10.1387\/ijdb.170230zk","article-title":"Amphioxus photoreceptors - Insights into the evolution of vertebrate opsins, vision and circadian rhythmicity.","volume":"61","author":"Pergner","year":"2017","journal-title":"Int. J. Dev. Biol."},{"key":"B26","doi-asserted-by":"publisher","first-page":"705","DOI":"10.3389\/fcell.2020.00705","article-title":"Molecular Fingerprint of Amphioxus Frontal Eye Illuminates the Evolution of Homologous Cell Types in the Chordate Retina.","volume":"8","author":"Pergner","year":"2020","journal-title":"Front. Cell Dev. Biol."},{"key":"B27","doi-asserted-by":"publisher","first-page":"49","DOI":"10.1016\/0306-9877(92)90014-4","article-title":"A review of in vivo studies of porphyrins and unexpected fluorescences. An interpretation of the results.","volume":"37","author":"Plus","year":"1992","journal-title":"Med. Hypotheses"},{"key":"B28","first-page":"159","article-title":"Some considerations on the fine structure of rhabdomeric photoreceptors in the amphioxus, Branchiostoma lanceolatum (cephalochordata).","volume":"32","author":"Ruiz","year":"1991","journal-title":"J. Hirnforsch."},{"key":"B29","doi-asserted-by":"publisher","first-page":"105","DOI":"10.1111\/j.1463-6395.1995.tb00986.x","article-title":"Embryos and Larvae of a Lancelet, Branchiostoma floridae, from Hatching through Metamorphosis: growth in the Laboratory and External Morphology.","volume":"76","author":"Stokes","year":"1995","journal-title":"Acta Zool."},{"key":"B30","doi-asserted-by":"publisher","first-page":"1709","DOI":"10.1038\/nprot.2015.085","article-title":"Advanced CUBIC protocols for whole-brain and whole-body clearing and imaging.","volume":"10","author":"Susaki","year":"2015","journal-title":"Nat. Protoc."},{"key":"B31","doi-asserted-by":"publisher","first-page":"15383","DOI":"10.1073\/pnas.1207580109","article-title":"Molecular analysis of the amphioxus frontal eye unravels the evolutionary origin of the retina and pigment cells of the vertebrate eye.","volume":"109","author":"Vopalensky","year":"2012","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B32","doi-asserted-by":"publisher","first-page":"122","DOI":"10.1139\/z04-163","article-title":"The nervous system of amphioxus: structure, development, and evolutionary significance.","volume":"83","author":"Wicht","year":"2005","journal-title":"Can. J. Zool."},{"key":"B33","doi-asserted-by":"publisher","first-page":"183","DOI":"10.1038\/044202a0","article-title":"The later larval development of amphioxus.","volume":"32","author":"Willey","year":"1891","journal-title":"J. Cell Sci."},{"key":"B34","doi-asserted-by":"publisher","DOI":"10.1038\/051433a0","author":"Willey","year":"1894","journal-title":"Amphioxus And The Ancestry Of The Vertebrates."},{"key":"B35","doi-asserted-by":"publisher","first-page":"1012","DOI":"10.2108\/zsj.25.1012","article-title":"The lancelet and ammocoete mouths.","volume":"25","author":"Yasui","year":"2008","journal-title":"Zoolog. Sci."},{"key":"B36","doi-asserted-by":"publisher","first-page":"465","DOI":"10.1002\/jmor.20228","article-title":"Development of oral and branchial muscles in lancelet larvae of Branchiostoma japonicum.","volume":"275","author":"Yasui","year":"2014","journal-title":"J. Morphol."},{"key":"B37","doi-asserted-by":"publisher","first-page":"28350","DOI":"10.1038\/srep28350","article-title":"The evolution of genes encoding for green fluorescent proteins: insights from cephalochordates (amphioxus).","volume":"6","author":"Yue","year":"2016","journal-title":"Sci. Rep."}],"container-title":["Frontiers in Cell and Developmental Biology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2021.702986\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,7,26]],"date-time":"2021-07-26T12:28:30Z","timestamp":1627302510000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fcell.2021.702986\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,26]]},"references-count":37,"alternative-id":["10.3389\/fcell.2021.702986"],"URL":"https:\/\/doi.org\/10.3389\/fcell.2021.702986","relation":{},"ISSN":["2296-634X"],"issn-type":[{"type":"electronic","value":"2296-634X"}],"subject":[],"published":{"date-parts":[[2021,7,26]]},"article-number":"702986"}}