ABSTRACTNodal signaling plays crucial roles in vertebrate developmental processes such as endoderm and mesoderm formation, and axial patterning events along the anteroposterior, dorsoventral and left-right axes. In echinoderms, Nodal plays an essential role in the establishment of the dorsoventral axis and left-right asymmetry, but not in endoderm or mesoderm induction. In protostomes, Nodal signaling appears to be involved only in establishing left-right asymmetry. Hence, it is hypothesized that Nodal signaling has been co-opted to pattern the dorsoventral axis of deuterostomes and for endoderm, mesoderm formation as well as anteroposterior patterning in chordates. Hemichordata, together with echinoderms, represent the sister taxon to chordates. In this study, we analyze the role of Nodal signaling in the indirect developing hemichordate Ptychodera flava. In particular, we show that during gastrulation nodal transcripts are detected in a ring of cells at the vegetal pole that gives rise to endomesoderm and in the ventral ectoderm at later stages of development. Inhibition of Nodal function disrupts dorsoventral fates and also blocks formation of the larval mesoderm. Interestingly, molecular analysis reveals that only mesodermal, apical and ventral gene expression is affected while the dorsal side appears to be patterned correctly. Taken together, this study suggests that the co-option of Nodal signaling in mesoderm formation and potentially in anteroposterior patterning has occurred prior to the emergence of chordates and that Nodal signaling on the ventral side is uncoupled from BMP signaling on the dorsal side, representing a major difference from the molecular mechanisms of dorsoventral patterning events in echinoderms.