Generation of a prop1 knockin 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 knockin 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.