A study in 2006 showed that tissue geometry determines the site of branching morphogenesis (Nelson et al., 2006). Using a micropatterning approach to control the initial 3D structure of mouse mammary epithelial tubules, it was determined that tubules dictate the position of the branches by defining the local concentration of TGF-β that acts in this context as an inhibitory morphogen. Subsequently, a modified 3D culture system for primary mouse mammary organoids using laminin-rich ECM in 96-well plates to culture the organoids was established (Fata et al., 2007). This new system that enables the simultaneous examination of multiple treatments allowed the authors to establish that the interplay between growth factors, their spatial localization, and the duration of their activation as well as downstream effectors cooperate and regulate whether mammary branches initiate and/or elongate. To assess in real time the cellular behavior that leads to branching morphogenesis, the system described above was used to carry out long-term confocal time-lapse analysis (Ewald et al., 2008; Huebner et al., 2016). The behavior of both the luminal and the myoepithelial cells was followed in real time, showing that mammary ducts elongate through a distinct type of collective epithelial migration, with no leading cell extensions or
