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

Cowin P (2013) Gli Activity Is Critical at Multiple Stages of Embryonic Mammary and Nipple Development. PLoS ONE 8(11): e79845. doi:10.1371/journal.pone.0079845 Gli Activity Is Critical at Multiple Stages of Embryonic Mammary and Nipple Development Anupama Chandramouli 0 Sarah J. Hatsell 0 Alicia Pinderhughes 0 Lisa Koetz 0 Pamela Cowin 0 Vladimir V. Kalinichenko, Cincinnati Children's Hospital Medical Center, United States of America 0 1 Department of Cell Biology, New York University School of Medicine , New York , New York, United States of America, 2 The Ronald O. Perelman Department of Dermatology, New York University School of Medicine , New York, New York , United States of America Gli3 is a transcriptional regulator of Hedgehog (Hh) signaling that functions as a repressor (Gli3R) or activator (Gli3A) depending upon cellular context. Previously, we have shown that Gli3R 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 Gli3xt/xt mice. Importantly, mice genetically manipulated to misactivate Hh signaling displayed the same phenotypic spectrum demonstrating that the repressor function of Gli3R 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 Gli3R is critical for early placodal patterning and later mammary mesenchyme specification whereas positive Hh signaling occurs during nipple development. - Funding: This work was supported by DOD BCRP IDEA Awards BC093088 (PC), BC074763 (SH/PC), DOD BCRP Fellowship BC112418 (AC), Susan G. Komen Foundation BCTR0504557 (PC), NIH-F31CA130137 (AP). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Mammary development becomes apparent in mice around E10.5 with expression of Wnt10b in mammary lines between the fore- and hind-limbs and in axillary and inguinal streaks [1,2,3]. Between E10.5 and E11.5, influx of epithelial cells towards and along these mammary lines and streaks leads to the formation of five pairs of placodes [4]. Analyses of knock-out mice and of human syndromes involving loss of mammary rudiments (MRs) or abnormal nipple number have identified more than a dozen factors essential for early mammary placodal development [3,5,6,7,8,9,10,11]. Among these factors, MR#3 formation depends upon reciprocal antagonism between ventrally expressed Bmp4 and dorsal Tbx3 [12]. At ,E12.5 the placodes form elevated buds. These buds sink below the periderm ,E13.5 to form bulb-like structures, which induce underlying fibroblasts to become mammary mesenchyme [13,14]. In females proliferation beginning at E15E16, causes the mammary bulbs to sprout, penetrate the underlying developing fat-pad, and branch to form a small ductal tree (,E18) [11,13,14,15,16]. The mammary mesenchyme in turn signals to the overlying epidermis to suppress hair follicles and form the nipple sheath [11,13,14,15,16]. In male embryos, intrinsic androgen response within the mesenchyme leads to atrophy of the buds [3,9,17,18,19,20]. The Hedgehog (Hh) pathway plays a central role in the patterning and proliferation of many tissues, and its requirement in epidermal appendages, such as hair follicles and teeth, has been particularly well documented [21,22,23,24,25]. The mammalian Hh ligands, Sonic (Shh), Indian (Ihh) and Desert (Dhh), bind to twelve-pass transmembrane receptors Patched (Ptch1, Ptch2) on neighboring cells [26,27]. This event relieves the seven-pass transmembrane protein smoothened (Smo) from Ptch-mediated repression and generates signals that are transduced by the Gli family of transcriptional activators and repressors (Gli1-3) [27,28,29]. Downstream Gli target genes, Ptch and Hhip (Hedgehog interacting protein), together with molecules acting at the level of ligand-binding such as cell surface bound Ig/ fibronectin family members Cdo and Boc provide feedback mechanisms at various levels to keep the pathway in check [30]. Hh target gene expression is determined by the ratio of activator to repressor (GliA: GliR) forms of Gli proteins [31,32,33]. Gli2 is expressed independently of Hh signals in a functionally inactive form (Gli2R) but becomes processed in response to Hh signals into an activator (Gli2A) that initiates Hh target gene transcription [34,35,36,37]. Gli1 is transcribed in a strictly Hh-dependent manner and once expressed, constitutively activates Hh target genes, including itself, and is considered to be a non-essential pathway amplifier [38,39,40,41,42]. These features make it a useful and reliable indicator of Hh pathway activation [31,37]. Gli3 functions as a transcriptional activator (Gli3A) or repressor (Gli3R) depending on the cellular context [31,43]. In the absence of Hh signals, Gli3A is proteolytically processed into Gli3R. Hh signals prevent this proteolytic conversion and also transcriptionally downregulate Gli3 [27,35,43,44,45]. Most tissues maintain a specific GliR:GliA ratio by feedback mechanisms regulating downstream target gene expression. The processing of Gli proteins occurs within the primitive vestigial organelle, primary cilium [46]. Intraflagellar transport proteins (Ift) associate with kinesins or dyneins and are responsible for the formation and maintenance of primary cilia [47,48]. Although hair follicles and mammary glands share many local inductive pathways, these appendages undergo strikingly different responses to Hh signaling [21,22,24,49,50,51,52,53]. Hair and teeth require Hh signaling for downgrowth. We have shown that Gli3-mediated repression of Hh signaling is essential for the formation of MR#3 and #5 [51]. Loss of Gli3 exerts milder effects on the development of remaining MRs. However the molecular consequences of Gli3 action and whether it functions as an activator or repressor of Hh signaling or via Hh-independent functions at later stages have not been addressed. Here we have investigated the effects of Gli3 inactivation on factors involved early in the specification of MR#3 and found that Gli3 is required for the correct patterning of Bmp4 and Tbx3. We further show that later in embryonic mammary developm (...truncated)