Quantitative imaging reveals real-time Pou5f3–Nanog complexes driving dorsoventral mesendoderm patterning in zebrafish

Formation of the three embryonic germ layers is a fundamental developmental process that initiates differentiation. How the zebrafish pluripotency factor Pou5f3 (homologous to mammalian Oct4) drives lineage commitment is unclear. Here, we introduce fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy to assess the formation of Pou5f3 complexes with other transcription factors in real-time in gastrulating zebrafish embryos. We show, at single-cell resolution in vivo, that Pou5f3 complexes with Nanog to pattern mesendoderm differentiation at the blastula stage. Later, during gastrulation, Sox32 restricts Pou5f3-Nanog complexes to the ventrolateral mesendoderm by binding Pou5f3 or Nanog in prospective dorsal endoderm. In the ventrolateral endoderm, the Elabela / Aplnr pathway limits Sox32 levels, allowing the formation of Pou5f3-Nanog complexes and the activation of downstream BMP signaling. This quantitative model shows that a balance in the spatiotemporal distribution of Pou5f3-Nanog complexes, modulated by Sox32, regulates mesendoderm specification along the dorsoventral axis.