Generation of a prop1 knock-in zebrafish enables single-cell transcriptomics of early pituitary development.

The adenohypophyseal placode is a developmental structure that is conserved among vertebrates and gives rise to the anterior pituitary. The earliest steps of its formation remain poorly understood. Here, we present a powerful new genetic tool: a zebrafish GAL4 knock-in reporter line for prop1. The targeted integration disrupts prop1 function, generating the first prop1-/- zebrafish, which exhibits dwarfism, impaired sexual maturation, and pigmentation defects. The generation of this new reporter line enabled the study of early pituitary development at single-cell resolution, using both scRNA-seq and live imaging. We identified epcam/Epcam as a conserved epithelial marker of the adenohypophysis in both zebrafish and mouse. We further studied transcriptional programs underlying endocrine lineage specification, discovering several previously unrecognized markers for hormone-producing lineages, including tfcp2l1, as a likely regulator of lactotrope differentiation. Cell-cell interaction analyses indicated Notch, Fgf, and Slit/Robo signaling as key regulators of progenitor maintenance and differentiation. Overall, this work provides the first single-cell atlas of early zebrafish pituitary development, a robust new genetic tool to study fundamental mechanistic insights into pituitary lineage specification and differentiation.