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Here we describe the generation of a Netrin-1 reporter chicken line (<jats:italic>NTN1-T2A-eGFP<\/jats:italic>) by targeting green fluorescent protein into the <jats:italic>NTN1<\/jats:italic> locus using CRISPR\/Cas9 methodology. Our strategy gave 100% transmission of heterozygous (<jats:italic>NTN1<\/jats:italic>\n            <jats:sup>\n              <jats:italic>T2A\u2009\u2212\u2009eGFP<\/jats:italic>\/+<\/jats:sup>) embryos in which GFP localisation faithfully replicated endogenous <jats:italic>NTN1<\/jats:italic> expression in the optic fissure and neural tube floorplate. Furthermore, all <jats:italic>NTN1<\/jats:italic>\n            <jats:sup>\n              <jats:italic>T2A\u2009\u2212\u2009eGFP<\/jats:italic>\/+<\/jats:sup> embryos and hatched birds appeared phenotypically normal. We applied this resource to a pertinent developmental context - coloboma is a structural eye malformation characterised by failure of epithelial fusion during optic fissure closure (OFC) and <jats:italic>NTN1<\/jats:italic> is specifically expressed in fusion pioneer cells at the edges of the optic fissure. We therefore optimised the isolation of GFP expressing cells from embryonic <jats:italic>NTN1<\/jats:italic>\n            <jats:sup>\n              <jats:italic>T2A\u2009\u2212\u2009eGFP<\/jats:italic>\/+<\/jats:sup> eyes using spectral fluorescence cell-sorting and applied transcriptomic profiling of pioneer cells, which revealed multiple new OFC markers and novel pathways for developmental tissue fusion and coloboma. This work provides a novel fluorescent <jats:italic>NTN1<\/jats:italic> chicken reporter line with broad experimental utility and is the first to directly molecularly characterise pioneer cells during OFC.<\/jats:p>","DOI":"10.1038\/s41598-025-94589-8","type":"journal-article","created":{"date-parts":[[2025,3,24]],"date-time":"2025-03-24T16:11:07Z","timestamp":1742832667000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["A stable NTN1 fluorescent reporter chicken reveals cell specific molecular signatures during optic fissure closure"],"prefix":"10.1038","volume":"15","author":[{"given":"Brian","family":"Ho Ching Chan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Holly","family":"Hardy","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Teresa","family":"Requena","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Amy","family":"Findlay","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jason","family":"Ioannidis","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dominique","family":"Meunier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria","family":"Toms","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mariya","family":"Moosajee","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Raper","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mike J.","family":"McGrew","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1091-5100","authenticated-orcid":false,"given":"Joe","family":"Rainger","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,3,24]]},"reference":[{"key":"94589_CR1","doi-asserted-by":"publisher","first-page":"2153","DOI":"10.1242\/dev.044529","volume":"138","author":"K Lai Wing Sun","year":"2011","unstructured":"Lai Wing Sun, K., Correia, J. 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