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The undifferentiated bipotential NMP state is maintained when both Wnt signaling is active and Sox2 is present. We used transgenic zebrafish reporter lines to live-image both Wnt activity and Sox2 levels in NMPs and observed a unique cellular ratio in NMPs compared to NMP-derived mesoderm or neural tissue. We used this unique signature to identify the previously unknown anatomical position of a progenitor population that gives rise to midline tissues of the floor plate of the spinal cord and the mesodermal notochord. Thus, quantification of the active Wnt signaling to Sox2 ratio can be used to predict and identify cells with neuromesodermal potential. We also developed the auxin-inducible 2 degron system for use in zebrafish to test the temporal role that Sox2 plays during midline formation. We found that ectopic Sox2 in the presence of Wnt activity holds cells in the undifferentiated floor plate\/notochord progenitor state, and that degradation of the ectopic Sox2 is required for cells to adopt a notochord fate.<\/jats:p>","DOI":"10.1242\/dev.204661","type":"journal-article","created":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T16:33:41Z","timestamp":1760114021000},"source":"Crossref","is-referenced-by-count":6,"title":["The ratio of Wnt signaling activity to Sox2 transcription factor levels predicts neuromesodermal fate potential"],"prefix":"10.1242","volume":"152","author":[{"given":"Robert M.","family":"Morabito","sequence":"first","affiliation":[{"name":"Stony Brook University 1 Department of Biochemistry and Cell Biology , , Stony Brook, NY 11733 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6297-2321","authenticated-orcid":false,"given":"David","family":"Tatarakis","sequence":"additional","affiliation":[{"name":"University of California 2 Department of Developmental and Cell Biology , , Irvine, CA 92697 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ryan","family":"Swick","sequence":"additional","affiliation":[{"name":"Stony Brook University 1 Department of Biochemistry and Cell Biology , , Stony Brook, NY 11733 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3255-6738","authenticated-orcid":false,"given":"Samantha","family":"Stettnisch","sequence":"additional","affiliation":[{"name":"Stony Brook University 1 Department of Biochemistry and Cell Biology , , Stony Brook, NY 11733 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4360-6729","authenticated-orcid":false,"given":"Pavan K.","family":"Nayak","sequence":"additional","affiliation":[{"name":"University of California 2 Department of Developmental and Cell Biology , , Irvine, CA 92697 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Thomas F.","family":"Schilling","sequence":"additional","affiliation":[{"name":"University of California 2 Department of Developmental and Cell Biology , , Irvine, CA 92697 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9536-7790","authenticated-orcid":false,"given":"Julia A.","family":"Horsfield","sequence":"additional","affiliation":[{"name":"Dunedin School of Medicine, University of Otago 3 Department of Pathology , , Dunedin 9016 ,","place":["New Zealand"]},{"name":"The Maurice Wilkins Centre for Biodiscovery, The University of Auckland 4 , Auckland 1010 ,","place":["New Zealand"]},{"name":"Genetics Otago Research Centre, University of Otago 5 , Dunedin 9016 ,","place":["New Zealand"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5474-4492","authenticated-orcid":false,"given":"Benjamin L.","family":"Martin","sequence":"additional","affiliation":[{"name":"Stony Brook University 1 Department of Biochemistry and Cell Biology , , Stony Brook, NY 11733 ,","place":["USA"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"237","published-online":{"date-parts":[[2025,11,17]]},"reference":[{"key":"2025111709525434000_DEV204661C1","doi-asserted-by":"publisher","first-page":"e63265","DOI":"10.7554\/eLife.63265","article-title":"Visualizing the metazoan proliferation-quiescence decision in vivo","volume":"9","author":"Adikes","year":"2020","journal-title":"eLife"},{"key":"2025111709525434000_DEV204661C2","doi-asserted-by":"publisher","first-page":"1397","DOI":"10.1242\/dev.125.8.1397","article-title":"Promoting notochord fate and repressing muscle development in zebrafish axial mesoderm","volume":"125","author":"Amacher","year":"1998","journal-title":"Development"},{"key":"2025111709525434000_DEV204661C3","doi-asserted-by":"publisher","first-page":"e1005967","DOI":"10.1371\/journal.pgen.1005967","article-title":"Competition between jagged-notch and endothelin1 signaling selectively restricts cartilage formation in the zebrafish upper face","volume":"12","author":"Barske","year":"2016","journal-title":"PLoS Genet."},{"key":"2025111709525434000_DEV204661C4","doi-asserted-by":"publisher","first-page":"633","DOI":"10.1038\/s41556-022-00910-2","article-title":"Sox2 levels regulate the chromatin occupancy of WNT mediators in epiblast progenitors responsible for vertebrate body formation","volume":"24","author":"Blassberg","year":"2022","journal-title":"Nat. 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