Uniaxial force modifies the length of the mammary ductal network and the orientation of ducts during pubertal development: Findings from computational modeling and laboratory experiments

Abstract

The orientation of the epithelium ducts determines the overall shape of the ductal network in the mammary gland, which in turn impacts the efficiency of the delivery of milk through the ducts to breastfeeding infants. However, how the orientations of the ducts are specified is not well understood. Cell-cell and cell-extracellular matrix (ECM) interactions perturb the tissue mechanical environment and influence mechanical signaling, which in turn regulates cell migration, tissue organization, and morphogenesis. This study examined if an applied force that perturbs the tissue mechanical environment can regulate the orientation of the epithelium ducts during puberty, in vivo. Uniaxial forces were applied continuously to the right abdominal number four mouse mammary glands (n=10; TEN) at 5-7 weeks of age, that is, during pubertal formation of the epithelial ductal network. The uniaxial force was applied by pulling and adhering the skin around the nipple of the right abdominal number four mammary gland. This pulling force on the right gland made the left abdominal number four gland also experience a contralateral (CONTRA) force. Mammary glands from litter mates were dissected at 3 weeks, 5 weeks, and 7 weeks of unmanipulated mice to serve as controls. Following dissection, whole mounts were prepared by carmine alum staining and panoramic images were captured under light microscopy. The ductal network in the images were skeletonized and straighten along the longitudinal midline to accurately capture the length of the ductal network without bias from its curvature. Findings from using the Measure tool in ImageJ version 1.54j indicated that the length of the ductal network was increased in the TEN and CONTRA mammary glands compared to control (P < 0.05). Analysis of the images using OrientationJ version 16.01.2018, an ImageJ plug-in, indicated that the orientation of ducts in the TEN and CONTRA mammary glands where altered compared to control (P < 0.05). Although the ductal network was longer in the TEN and CONTRA glands compared to control, there was no significant difference in the total cross-sectional area. In-silico simulations of the ductal network formation using a branching and annihilating random walk model predicted that the increased length of the ductal network may have resulted from changes in the orientation of the epithelium ducts. Thus it is likely that mechanical forces regulate the orientation of ductal branches in vivo.