Nodal and <i>churchill1</i> position the expression of a notch ligand during <i>Xenopus</i> germ layer segregation.

In vertebrates, Nodal signaling plays a major role in endomesoderm induction, but germ layer delimitation is poorly understood. In avian embryos, the neural/mesoderm boundary is controlled by the transcription factor CHURCHILL1, presumably through the repressor ZEB2, but there is scarce knowledge about its role in other vertebrates. During amphibian gastrulation, Delta/Notch signaling refines germ layer boundaries in the marginal zone, but it is unknown the place this pathway occupies in the network comprising Churchill1 and Nodal. Here, we show that <i>Xenopus churchill1</i> is expressed in the presumptive neuroectoderm at mid-blastula transition and during gastrulation, upregulates <i>zeb2</i>, prevents <i>dll1</i> expression in the neuroectoderm, and favors neuroectoderm over endomesoderm development. Nodal signaling prevents <i>dll1</i> expression in the endoderm but induces it in the presumptive mesoderm, from where it activates Notch1 and its target gene <i>hes4</i> in the non-involuting marginal zone. We propose a model where Nodal and Churchill1 position Dll1/Notch1/Hes4 domains in the marginal zone, ensuring the delimitation between mesoderm and neuroectoderm.