Noncanonical Wnt/Ror2 Signaling Regulates Basal Cell Fidelity and Branching Morphogenesis in the Mammary Gland

The mammary gland epithelium relies on a delicate balance between basal and luminal cell lineages to maintain tissue homeostasis and enable proper development. While the role of canonical Wnt signaling in mammary biology is well-established, the contribution of noncanonical Wnt signaling to lineage identity has remained unclear. Noncanonical Wnt pathways are primarily associated with morphogenesis, cytoskeletal regulation, and cell migration, but whether they are required for maintaining epithelial cell fate remains largely unexplored. Here, we demonstrate that the noncanonical Wnt receptor Ror2 is expressed in both basal and luminal lineages, yet selectively maintained in basal cells throughout development, suggesting a lineage-specific function. Using a p63CreERT2/+ lineage-specific mouse model, we show that Ror2 deletion in basal epithelial cells enhances secondary and tertiary branching while driving a basal-to-luminal fate transition, marked by downregulation of basal markers (K14, K5) and upregulation of luminal markers (K8, K18, ER). Mechanistically, Ror2 loss disrupts RhoA-ROCK1-YAP1 signaling, leading to cytoskeletal reorganization, chromatin remodeling, and increased accessibility at luminal regulatory loci. Notably, ROCK1 inhibition phenocopies Ror2 loss, reinforcing the critical role of the RhoA-ROCK1 axis in basal cell maintenance. These findings provide direct genetic and mechanistic evidence that noncanonical Wnt signaling is essential for maintaining basal lineage fidelity, offering new insights into the mechanisms regulating epithelial plasticity. Given the fundamental importance of lineage stability in epithelial homeostasis, our results suggest that disruptions in Wnt/Ror2 signaling may contribute to aberrant fate transitions relevant to breast cancer progression.