Tissue Geometry Determines Sites of

land, do not allow for a precise quantitative understanding of how spatial positioning is determined. Given that the mammary ductal network branches out from preexisting epithelial tubules, we hypothesized that the position of cells within a tubule might provide contextual information to instruct branch site initiation. To define the role of positional context, we developed a three-dimensional (3D) micropatterned assay for mammary epithelial branching morphogenesis that allowed us to mimic the mammary rudiment by controlling the initial geometry of tubules and to quantify the positions at which they branched. We engineered epithelial tubules of defined geometry by embedding functionally normal mouse mammary epithelial (EpH4) cells in cavities of collagen gel generated by molding unpolymerized collagen I around a patterned elastomeric stamp (Fig. 1A) (6). Embedded epithelial cells formed hollow tubules (7) conforming to the size and shape of the collagen cavities (Fig. 1, B and C, an