3D spatial organization of heterogeneous nkx2.5+ progenitors in the zebrafish heart field pre-patterns cardiovascular development

Nkx2.5+ progenitor cells are specified in the bilateral zebrafish heart field, which contributes to multiple cardiovascular derivatives. After specification, the field physically separates into a cardiac fraction that migrates to the midline to form ventricular and atrial myocardium, a pharyngeal fraction that is sequestered in the pharyngeal arches before generating head muscles, the outflow tract, and pharyngeal arch arteries, and a lateral fraction that becomes inflow atrial and pacemaker cardiomyocytes as well as pericardium. Whether these progenitors are transcriptionally distinct and regionally organized has remained unclear. Here, we demonstrate that the heart field is composed of heterogeneous nkx2.5+ progenitor populations that are arranged according to their migratory paths and end fates. Through prospective fate mapping and mutant analyses, we define the derivatives of each subgroup. Our data provide a 3D blueprint of heart field organization, offering insights into cardiovascular development and identifying vulnerabilities contributing to congenital heart disease.