The varying pathways of mammary gland development across species and evolutionary history are underexplored, largely due to a lack of model systems. Recent progress in organoid technology holds the promise of enabling in-depth studies of the developmental adaptations that have occurred throughout the evolution of different species, fostering beneficial phenotypes. The practical application of this technology for mammary glands has been mostly confined to rodents and humans. In the current study, we have successfully created next-generation 3D mammary gland organoids from eight eutherian mammals and the first branched organoid of a marsupial mammary gland. Using mammary organoids, we identified a role for ROCK protein in regulating branching morphogenesis, a role that manifests differently in organoids from different mammals. This finding demonstrates the utility of the 3D organoid model for understanding the evolution and adaptations of signaling pathways. These achievements highlight the potential for organoid models to expand our understanding of mammary gland biology and evolution, and their potential utility in studies of lactation or breast cancer.

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