Sox9 inhibits Activin A to promote biliary maturation and branching morphogenesis.

Intrahepatic bile duct (IHBD) development produces a morphologically heterogeneous network of large "ducts" and small "ductules" by adulthood. IHBD formation is closely linked to developmental specification of biliary epithelial cells (BECs) starting as early as E13.5, but mechanisms regulating differential IHBD morphology remain poorly understood. Here, we show that duct and ductule development has distinct genetic requirements, with Sox9 required to form the developmental precursors to peripheral ductules in adult livers. By optimizing large-volume IHBD imaging, we find that IHBDs emerge as a homogeneous webbed structure by E15.5 and undergo morphological maturation through 2 weeks of age. Developmental knockout of Sox9 leads to decreased postnatal branching morphogenesis, resulting in adult IHBDs with normal ducts but significantly fewer ductules. In the absence of Sox9, BECs fail to mature and exhibit elevated TGF-β signaling and Activin A. Exogenous Activin A is sufficient to induce developmental gene expression and morphological defects in wild-type BEC organoids, while early postnatal inhibition of Activin A in vivo rescues IHBD morphogenesis in the absence of Sox9. Our data demonstrate that proper IHBD architecture relies on inhibition of Activin A by Sox9 to promote ductule morphogenesis, defining regulatory mechanisms underlying morphological heterogeneity.