Gli Activity Is Critical at Multiple Stages of Embryonic Mammary and Nipple Development

Gli3 is a transcriptional regulator of Hedgehog (Hh) signaling that functions as a repressor (Gli3^R) or activator (Gli3^A) depending upon cellular context. Previously, we have shown that Gli3^R is required for the formation of mammary placodes #3 and #5. Here, we report that this early loss of Gli3 results in abnormal patterning of two critical regulators: Bmp4 and Tbx3, within the presumptive mammary rudiment (MR) #3 zone. We also show that Gli3 loss leads to failure to maintain mammary mesenchyme specification and loss of epithelial Wnt signaling, which impairs the later development of remaining MRs: MR#2 showed profound evagination and ectopic hairs formed within the presumptive areola; MR#4 showed mild invagination defects and males showed inappropriate retention of mammary buds in Gli3^xt/xt mice. Importantly, mice genetically manipulated to misactivate Hh signaling displayed the same phenotypic spectrum demonstrating that the repressor function of Gli3^R is essential during multiple stages of mammary development. In contrast, positive Hh signaling occurs during nipple development in a mesenchymal cuff around the lactiferous duct and in muscle cells of the nipple sphincter. Collectively, these data show that repression of Hh signaling by Gli3^R is critical for early placodal patterning and later mammary mesenchyme specification whereas positive Hh signaling occurs during nipple development.