To determine the importance of Hh signaling in vivo, two mouse lines representing either a gain-of-function or a loss-of-function will be utilized. One line is transgenic for constitutively active Smoothened (SMO) protein fused to yellow fluorescent protein (YFP), whose expression is positively regulated by Cre recombinase activity. SMO is the Hh signaling effector and the limiting component of the signaling network. Primary mammary epithelial cells (MECs) obtained from these mice have been infected with Adenovirus expressing Cre recombinase (Adenovirus-Cre) and subsequently transplanted into cleared fat pads of immune compromised mice. The second line is a Cre-dependent (“floxed”) SMO conditional knockout, genetically tagged with a Cre-dependent ROSA26-lacZ reporter allele. As above, primary MECs will be obtained, infected with Adenovirus-Cre and transplanted into cleared fat pads. In additional experiments, both lines will be crossed to an MMTV-Cre line to induce recombination in all the tissues in which the mouse mammary tumor virus (MMTV) promoter is active, primarily the mammary and salivary glands. Finally, nipple injections of the Adenovirus-Cre will also be performed in both lines to activate or delete SMO in a subset of ductal mammary epithelial cells.  Analysis of these experiments on these two lines will allow for in vivo observation of the effects of activating and repressing Hh signaling on mammary gland outgrowth, branching, and stem cell behavior.