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However, the functional characteristics of\n                    <jats:italic>in vitro<\/jats:italic>\n                    SOX2\/TBXT co-expressing cells remain unclear, confounding comparisons with\n                    <jats:italic>in<\/jats:italic>\n                    <jats:italic>vivo<\/jats:italic>\n                    data. To address this, we developed a dual\n                    <jats:italic>Sox2\/Tbxt<\/jats:italic>\n                    reporter mouse ESC line. SOX2\/TBXT reporter-positive cells emerge\n                    <jats:italic>in vitro<\/jats:italic>\n                    from pluripotent populations with dynamics that mirror their appearance in the embryo. Purified SOX2\/TBXT co-expressing populations can differentiate towards neurectoderm or mesoderm, including lateral mesoderm upon BMP stimulation. In gastruloids, quantitative live imaging shows that WNT or NOTCH inhibition rapidly leads to downregulation of TBXT expression and diminished axial extension. We show that clonally plated SOX2\/TBXT co-expressing cells are bipotent NMPs that can also self-propagate. By combining clonal analysis with mathematical inference, we identify two thresholds of TBXT and\/or SOX2 expression, switching clonal output from neural- to mesoderm-biased, and from mesoderm-biased to mesoderm-specified. Image analysis of embryonic NMPs supports a model whereby SOX2 and TBXT independently influence neuromesodermal differentiation. Thus, this\n                    <jats:italic>Sox2\/Tbxt<\/jats:italic>\n                    double reporter cell line highlights unsuspected heterogeneity in NMPs, and together with image analysis of embryonic SOX2\/TBXT levels, challenges the assumption that neuromesodermal fate choice is primarily governed by mutual antagonism between SOX2\/TBXT.\n                  <\/jats:p>","DOI":"10.1371\/journal.pbio.3003960","type":"journal-article","created":{"date-parts":[[2026,8,25]],"date-time":"2026-08-25T17:37:01Z","timestamp":1787679421000},"page":"e3003960","update-policy":"https:\/\/doi.org\/10.1371\/journal.pbio.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Brachyury expression levels predict lineage potential and axis-forming ability of in vitro-derived neuromesodermal progenitors"],"prefix":"10.1371","volume":"24","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7987-9286","authenticated-orcid":true,"given":"Anah\u00ed","family":"Binagui-Casas","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Gran\u00e9s","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alberto S.","family":"Ceccarelli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew","family":"French","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Filip J.","family":"Wymeersch","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rosa","family":"Portero Migueles","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"Annoh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yali","family":"Huang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eleni P.","family":"Karagianni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frederick C. 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