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• W2087028407 abstract "The Hedgehog signaling pathway regulates the development and function of numerous tissues and when mis-regulated causes tumorigenesis. To assess the role of a deregulated Hedgehog signaling pathway in the mammary gland we targeted the expression of the Hedgehog effector protein, GLI1, to mammary epithelial cells using a bigenic inducible system. A constitutively active Hedgehog signaling pathway resulted with 100% penetrance in an undifferentiated mammary lobuloalveolar network during pregnancy. GLI1-expressing transgenic females were unable to lactate and milk protein gene expression was essentially absent. The inability to lactate was permanent and independent of continued GLI1 transgene expression. An increased expression of the GLI1 response gene Snail coupled to reduced expression of E-cadherin and STAT5 in the transgenic mammary gland provides a likely molecular explanation, underlying the observed phenotypic changes. In addition, remodeling of the mammary gland after parturition was impaired and expression of GLI1 was associated with accumulation of cellular debris in the mammary ducts during involution, indicating a defect in the clearance of dead cells. Areas with highly proliferative epithelial cells were observed in mammary glands with induced expression of GLI1. Within such areas an increased frequency of cells expressing nuclear Cyclin D1 was observed. Taken together the data support the notion that correct regulation of Hedgehog signaling within the epithelial cell compartment is critical for pregnancy-induced mammary gland development and remodeling. The Hedgehog signaling pathway regulates the development and function of numerous tissues and when mis-regulated causes tumorigenesis. To assess the role of a deregulated Hedgehog signaling pathway in the mammary gland we targeted the expression of the Hedgehog effector protein, GLI1, to mammary epithelial cells using a bigenic inducible system. A constitutively active Hedgehog signaling pathway resulted with 100% penetrance in an undifferentiated mammary lobuloalveolar network during pregnancy. GLI1-expressing transgenic females were unable to lactate and milk protein gene expression was essentially absent. The inability to lactate was permanent and independent of continued GLI1 transgene expression. An increased expression of the GLI1 response gene Snail coupled to reduced expression of E-cadherin and STAT5 in the transgenic mammary gland provides a likely molecular explanation, underlying the observed phenotypic changes. In addition, remodeling of the mammary gland after parturition was impaired and expression of GLI1 was associated with accumulation of cellular debris in the mammary ducts during involution, indicating a defect in the clearance of dead cells. Areas with highly proliferative epithelial cells were observed in mammary glands with induced expression of GLI1. Within such areas an increased frequency of cells expressing nuclear Cyclin D1 was observed. Taken together the data support the notion that correct regulation of Hedgehog signaling within the epithelial cell compartment is critical for pregnancy-induced mammary gland development and remodeling. The Hedgehog (Hh) 2The abbreviations used are: Hh, hedgehog; STAT, signal transducers and activators of transcription; TRE, tetracycline responsive elements; WT, wild type; dpc, days of pregnancy; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMTV, murine mammary tumor virus; RT, reverse transcriptase; WAP, whey acidic protein; L1, lactation day 1; I14, involution day 14. 2The abbreviations used are: Hh, hedgehog; STAT, signal transducers and activators of transcription; TRE, tetracycline responsive elements; WT, wild type; dpc, days of pregnancy; PBS, phosphate-buffered saline; BrdUrd, bromodeoxyuridine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MMTV, murine mammary tumor virus; RT, reverse transcriptase; WAP, whey acidic protein; L1, lactation day 1; I14, involution day 14. signal transduction pathway controls a variety of developmental processes such as pattern formation, differentiation, proliferation, and organogenesis and when misregulated causes the formation of developmental defects and tumorigenesis. The molecular details of the Hh signaling pathway have been thoroughly investigated using genetic studies in Drosophila melanogaster (for review see Refs. 1Cohen Jr., M.M. Am. J. Med. Genet. A. 2003; 123: 5-28Crossref Scopus (375) Google Scholar and 2Hooper J.E. Scott M.P. Nat. Rev. Mol. Cell. Biol. 2005; 6: 306-317Crossref PubMed Scopus (675) Google Scholar). The hh signaling cascade starts by binding of hh to the receptor protein ptc, which abrogates its inhibitory effect on smoothened (smo), a 7-span transmembrane co-receptor protein. The active smo protein signals downstream to a microtubule-associated multiprotein complex resulting in a switch from generation of the proteolytically cleaved repressor form of the transcription factor ci to release of a full-length activator form of ci that enters the nucleus and activates target genes. The Hh signaling pathway is well conserved through evolution. However, it is far more complicated in vertebrates with three Hh genes, Sonic, Indian, and Desert (Shh, Ihh, and Dhh); two ptc genes (Ptch1 and Ptch2); and three ci homologues (Gli1, Gli2, and Gli3). In vertebrates, the Hh signal activates Gli2 initiating transcription of Hh target genes, including Ptch1 and Gli1 (3Bai C.B. Auerbach W. Lee J.S. Stephen D. Joyner A.L. Development. 2002; 129: 4753-4761Crossref PubMed Scopus (91) Google Scholar). Gli2 and Gli3 can be expressed in the absence of Hh signaling, whereas Gli1 is strictly dependent on Hh signaling for its expression, which makes Gli1 an excellent reporter of active Hh signaling (3Bai C.B. Auerbach W. Lee J.S. Stephen D. Joyner A.L. Development. 2002; 129: 4753-4761Crossref PubMed Scopus (91) Google Scholar, 4Bai C.B. Stephen D. Joyner A.L. Dev. Cell. 2004; 6: 103-115Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). Gli2 and Gli3 are bifunctional in that they like ci can be processed into a repressor form and function as transcriptional activators (5Wang Q.T. Holmgren R.A. Development. 2000; 127: 3131-3139Crossref PubMed Google Scholar). However, because Gli1 can replace Gli2 function in vivo, it is likely that Gli2 lacks a significant repressor function (6Aza-Blanc P. Lin H. Ruiz i Altaba A. Kornberg T.B. Development. 2000; 127: 4293-4301Crossref PubMed Google Scholar). Gli1 is apparently not proteolytically processed and is acting as a strong transcriptional activator (7Dai P. Akimaru H. Tanaka Y. Maekawa T. Nakafuku M. Ishii S. J. Biol. Chem. 1999; 274: 8143-8152Abstract Full Text Full Text PDF PubMed Scopus (435) Google Scholar, 8Lee J. Platt K.A. Censullo P. Ruiz i Altaba A. Development. 1997; 124: 2537-2552Crossref PubMed Google Scholar, 9Ruiz i Altaba A. Development. 1998; 125: 2203-2212Crossref PubMed Google Scholar). Remarkably, Gli1-/- mice are viable and appear normal, whereas Gli1/Gli2 double mutants have a more severe phenotype than Gli2 mutants indicating that Gli1 and Gli2 have overlapping functions (10Park H.L. Bai C. Platt K.A. Matise M.P. Beeghly A. Hui C.C. Nakashima M. Joyner A.L. Development. 2000; 127: 1593-1605Crossref PubMed Google Scholar). The Hh signaling pathway is essential for the development of numerous tissues and organs including many vertebrate ectodermal appendages that arise as a result of epithelial-mesenchymal interactions (11Ingham P.W. McMahon A.P. Genes Dev. 2001; 15: 3059-3087Crossref PubMed Scopus (2422) Google Scholar, 12McMahon A. Ingham P. Tabin C. Curr. Top. Dev. Biol. 2003; 53: 1-114Crossref PubMed Google Scholar). In analogy mammary gland development is dependent on epithelial-mesenchymal interactions and the Hh pathway is emerging as one important factor during mammary gland development (13Hatsell S.J. Cowin P. Development. 2006; 133: 3661-3670Crossref PubMed Scopus (84) Google Scholar, 14Lewis M.T. Ross S. Strickland P.A. Sugnet C.W. Jimenez E. Scott M.P. Daniel C.W. Development. 1999; 126: 5181-5193Crossref PubMed Google Scholar, 15Lewis M.T. Ross S. Strickland P.A. Sugnet C.W. Jimenez E. Hui C. Daniel C.W. Dev. Biol. 2001; 238: 133-144Crossref PubMed Scopus (78) Google Scholar, 16Moraes R.C. Zhang X. Harrington N. Fung J.Y. Wu M.F. Hilsenbeck S.G. Allred D.C. Lewis M.T. Development. 2007; 134: 1231-1242Crossref PubMed Scopus (149) Google Scholar, 17Veltmaat J.M. Relaix F. Le L.T. Kratochwil K. Sala F.G. van Veelen W. Rice R. Spencer-Dene B. Mailleux A.A. Rice D.P. Thiery J.P. Bellusci S. Development. 2006; 133: 2325-2335Crossref PubMed Scopus (93) Google Scholar). Development of the mouse mammary gland is initiated around day 10.5 of embryonic development with the formation of bilateral milk lines running between the fore and hind limbs. Epidermal cells within the milk line become columnar and multilayered, defining a ridge that protrudes above and below the plane of the single layered primitive epidermis. Five pair of placodes are formed at specific locations along the mammary line. Invaginations of cells within the placode into the underlying mesenchyme establish a bud, which elongates and gradually establish a rudimentary ductal tree. During puberty, body and cap cells of the terminal end buds are hormonally stimulated to differentiate and to form an open ductal tree that eventually fills the entire mammary fat pad. During pregnancy, hormonal stimulation alters the morphology of the mammary gland from a ductal to a more lobuloalveolar morphology via branching morphogenesis. At this point, lobuloalveolar progenitor cells in the ducts proliferate and form alveolar buds, which further differentiate into alveoli. After parturition, upon suckling, milk is released until suckling ceases and the gland undergoes involution. During this process most alveolar cells are committed to apoptosis as part of the gland remodeling process. However, a small portion of fully committed alveolar cells escape the involution process and function as alveolar progenitors during subsequent pregnancies (18Wagner K.U. Boulanger C.A. Henry M.D. Sgagias M. Hennighausen L. Smith G.H. Development. 2002; 129: 1377-1386Crossref PubMed Google Scholar). The Hh homologues Shh, Ihh, and Dhh are expressed in the mouse mammary gland (19Kouros-Mehr H. Werb Z. Dev. Dyn. 2006; 235: 3404-3412Crossref PubMed Scopus (164) Google Scholar, 20Michno K. Boras-Granic K. Mill P. Hui C.C. Hamel P.A. Dev. Biol. 2003; 264: 153-165Crossref PubMed Scopus (52) Google Scholar). Most likely, Ihh and Shh have redundant functions in mammary gland development during embryogenesis, since the embryonic mammary gland develops normally in Shh and Ihh knock-out mice (21Bitgood M. Shen L. McMahon A. Curr. Biol. 1996; 6: 298-304Abstract Full Text Full Text PDF PubMed Scopus (514) Google Scholar, 22Gallego M.I. Beachy P.A. Hennighausen L. Robinson G.W. Dev. Biol. 2002; 249: 131-139Crossref PubMed Scopus (46) Google Scholar). However, it is interesting to note that Ihh expression is regulated by progesterone, one of the key steroid hormones controlling ductal and alveolar development in the mammary gland (23Lee K. Jeong J. Kwak I. Yu Lanske B. Soegiarto D. Toftgard D. Tsai M. Tsai S. Lydon J. DeMayo F. Nat. Genet. 2006; 38: 1204-1209Crossref PubMed Scopus (205) Google Scholar, 24Matsumoto H. Zhao X. Das S.K. Hogan B.L. Dey S.K. Dev. Biol. 2002; 245: 280-290Crossref PubMed Scopus (148) Google Scholar, 25Takamoto N. Zhao B. Tsai S.Y. DeMayo F.J. Mol. Endocrinol. 2002; 16: 2338-2348Crossref PubMed Scopus (139) Google Scholar). The core components (Shh, Ihh, Dhh, Ptch1, Smo, Gli1, Gli2, Gli3) of the Hh signaling pathway have been reported to be expressed during essentially all stages of mammary gland development (13Hatsell S.J. Cowin P. Development. 2006; 133: 3661-3670Crossref PubMed Scopus (84) Google Scholar, 15Lewis M.T. Ross S. Strickland P.A. Sugnet C.W. Jimenez E. Hui C. Daniel C.W. Dev. Biol. 2001; 238: 133-144Crossref PubMed Scopus (78) Google Scholar, 19Kouros-Mehr H. Werb Z. Dev. Dyn. 2006; 235: 3404-3412Crossref PubMed Scopus (164) Google Scholar). Expression of Hh pathway components varies during the different phases of mammary gland development and is progressively elevated during pregnancy and a peak of expression is detected during lactation (13Hatsell S.J. Cowin P. Development. 2006; 133: 3661-3670Crossref PubMed Scopus (84) Google Scholar, 14Lewis M.T. Ross S. Strickland P.A. Sugnet C.W. Jimenez E. Scott M.P. Daniel C.W. Development. 1999; 126: 5181-5193Crossref PubMed Google Scholar, 19Kouros-Mehr H. Werb Z. Dev. Dyn. 2006; 235: 3404-3412Crossref PubMed Scopus (164) Google Scholar). Furthermore, the Hedgehog signaling components PTCH1, GLI1, and GLI2 are expressed in normal human mammary progenitor cells (26Liu S. Dontu G. Mantle I.D. Patel S. Ahn N.S. Jackson K.W. Suri P. Wicha M.S. Cancer Res. 2006; 66: 6063-6071Crossref PubMed Scopus (1035) Google Scholar). When it comes to studies of the role of Hh signaling during mammary gland development a complication resides in the fact that homozygous null mutations of several key network genes, including Shh, Ihh, Ptch1, Smo, Sufu, Gli2, and Gli3 are embryonic or perinatally lethal. However, hyperplastic changes appear in mammary glands from virgin heterozygous Ptch1+/- mice, in mice expressing constitutively active Smo in the mammary epithelium, in embryonic mammary transplants from Gli2 null mice, and in humanized fatpads of NOD-SCID mice transplanted with Gli2 overexpressing mammosphere initiating cells (14Lewis M.T. Ross S. Strickland P.A. Sugnet C.W. Jimenez E. Scott M.P. Daniel C.W. Development. 1999; 126: 5181-5193Crossref PubMed Google Scholar, 15Lewis M.T. Ross S. Strickland P.A. Sugnet C.W. Jimenez E. Hui C. Daniel C.W. Dev. Biol. 2001; 238: 133-144Crossref PubMed Scopus (78) Google Scholar, 16Moraes R.C. Zhang X. Harrington N. Fung J.Y. Wu M.F. Hilsenbeck S.G. Allred D.C. Lewis M.T. Development. 2007; 134: 1231-1242Crossref PubMed Scopus (149) Google Scholar, 26Liu S. Dontu G. Mantle I.D. Patel S. Ahn N.S. Jackson K.W. Suri P. Wicha M.S. Cancer Res. 2006; 66: 6063-6071Crossref PubMed Scopus (1035) Google Scholar). Furthermore, Gli3xt/xt mutants lack two pairs of mammary buds, which supports a role for Hh signaling during embryonic mammary gland development. In addition, loss of Gli3 expression induces Gli1 mis-expression in the mammary mesenchyme supporting the conclusion that during embryonic mammary gland formation the primary role for Gli3 is to repress Hh inducible target genes (13Hatsell S.J. Cowin P. Development. 2006; 133: 3661-3670Crossref PubMed Scopus (84) Google Scholar). In this report, we show that targeted expression of GLI1 in the mammary epithelial cell compartment results in a reduced mammary epithelial network and alveologenesis during pregnancy. Transgenic female mice are unable to lactate and on a molecular level, the lack of terminal development and differentiation is reflected by impaired milk (WAP, α-lactalbumin, and β-casein) gene expression. We propose that the effects observed are due to GLI1 induction of Snail coupled to reduced expression of E-cadherin and STAT5, which results in a reduced alveolar differentiation. Strikingly, in subsequent pregnancies, without induced GLI1 expression, the glands do not fully develop and the dams fail to lactate, showing profound and long lasting effects of a temporally restricted GLI1 expression. Moreover, the involution process is impaired in GLI1-induced mammary glands, which may explain the sustained defects in the multiparous mice with induced GLI1 expression only during the first pregnancy. In addition, an increase of Cyclin D1 expression and epithelial cell proliferation rate was observed, which frequently was associated with the formation of expanded clusters of epithelial cells. Transgenic Mice and Genotyping—A tetracycline-regulated GLI1 transgenic construct was made by subcloning a PCR amplified tetracycline responsive promoter consisting of five tetracycline (tet) responsive elements (TRE) and the minimal cytomegalovirus promoter region of pTRELuc (Clontech Inc.) in front of the human full-length GLI1 (human and mouse Gli1 show 85% similarity at the amino acid level). This construct was subcloned into the SalI/BamI-digested Bluescript KS(-) vector (pBSpA) harboring a 0.6-kb fragment of a rabbit β-globin intron and a 1.1-kb fragment containing two 3′-poly(A) signal sequences from the p5′BK5II plasmid, obtained from Dr. Jose Jorcano (CIEMAT, Madrid, Spain). The 3.6-kb fragment of GLI1 cDNA was cleaved with NheI/SnaBI and introduced into the corresponding site of the TRE/pBSA plasmid. The construct was verified by sequencing and tested for its tetracycline inducibility in a reporter-based assay in a 293TetTOn cell line purchased from Clontech. The 5.3-kb transgene was isolated by digestion with NotI/SalI and injected into pronuclei of fertilized [C57BL/6J × CBA] F2 oocytes. The putative founder transgenic mice were genotyped by PCR amplification of sequences specific for the TREGLI1 using tail genomic DNA and primers TREGLI1: R1 primer (5′-CGGTGTCTTCTATGGAGGTCAA-3′), F1 primer (5′-ACCCGGGTCGAGTAGGCGTGTA-3′). Founders were crossed with negative littermates to establish the F1 generation. The level of expression of GLI1 was studied in five transgenic lines by crossing TREGLI1 F1 mice with K5rtTA F1 mice, which carry a mutated form of the reverse tet activator gene downstream of the skin-specific keratin 5 (K5) promoter (27Ramirez A. Bravo A. Jorcano J.L. Vidal M. Differentiation. 1994; 58: 53-64PubMed Google Scholar). As a target line for studies in breast, one of the three TREGLI1 F1 lines that exhibited similar GLI1 expression levels in the skin and profound phenotype was chosen. This TREGLI1 F1 line was crossed with mice carrying the MMTVrtTA promoter upstream of the tet activator gene (28Gunther E.J. Belka G.K. Wertheim G.B. Wang J. Hartman J.L. Boxer R.B. Chodosh L.A. FASEB J. 2002; 16: 283-292Crossref PubMed Scopus (167) Google Scholar) and the offspring were used for the experiments. Doxycycline (2 mg/ml) and 5% sucrose was added to the drinking water and doxycycline treatment was continued until the animal was sacrificed if not stated otherwise. The bitransgenic mice were hemizygous for each transgene and compared with non-treated bigenic animals or WT-treated siblings. Genotyping of mice was performed by PCR and primers for TREGLI1 were as described above and for MMTVrtTA: pA primer (5′-ATCCGCACCCTTGATGACTCCG-3′) and pB primer (5′-GGCT ATCAACCAACACACTGCCAC-3′). All transgenic mice were generated within an SPF barrier facility according to local and national regulations, and experimental conditions were approved by the Stockholm South Animal Ethics Committee. Reverse Transcription-PCR—RNA was isolated using RNA-zolB (CRP Inc.) and random hexamer-primed complementary DNA (cDNA) was generated using the reverse transcription system (Promega). Primer pairs specific for Ptch1 (Ptch1.f 5′-GAATCCAGGCATCACCCACC-3′, Ptch1.B 5′-CCACGTCCTGCAGCTCAATG-3′), Gli1 (Gli1.a.F 5′-AGACCAGCAGCTGCACTGAA-3′), Gli1.b.B 5′-TGGCAGGTTGCACGTGGTC-3′), rtTA (rtTA-404.F 5′-TGACCTCCATAGAAGACACC-3′, Gli1-474.r 5′-GGGCCCTTTTTGGTGATTCA-3′), and Actin (actin.F 5′-GACAGGATGCAGAAGGAGAT-3′, actin.B 5′-TTGCTGATCCACATCTGCTG-3′) were used for analysis of mRNA levels. All experiments were independently repeated at least three times and no amplification was obtained without reverse transcriptase (data not shown). All cDNA products were resolved by electrophoresis using 1-4% agarose gels. Histological Analysis, Preparation of Mammary Whole Mounts, and TUNEL Assays—Gland fragments from different developmental stages (5 weeks, 10 weeks, 6.5 days of pregnancy (dpc), 18.5 dpc, lactation day 1 (L1, also termed parturition day), lactation day 2 (L2, 1 day after parturition), and involution day 14 (I14, 14 days after parturition)) were embedded in paraffin, sectioned, and hematoxylin/eosin stained. Mammary glands for whole mounts were spread on glass slides and fixed 4 h in Carnoy fixative. The tissue slides were sequentially rehydrated 15 min each in a graded ethanol (70, 50, 30, 10%, and H2O) and stained overnight (0.2% (w/v) carmine (Sigma), 0.5% (w/v) aluminum potassium sulfate (Sigma)). The slides were dehydrated 15 min each in a graded ethanol (70, 95, 100%) and Xylene and mounted with Pertex (Histolab). To detect apoptotic nuclei, paraformaldehyde-fixed paraffin sections were analyzed by terminal deoxynucleotidyl transferase digoxygenin nick-end labeling using the Apoptag kit (Chemicon) following the manufacturer's instructions. For quantitative analysis 1000 nuclei from three separate areas in three samples were counted. Immunohistochemical Analysis—Mammary glands were fixed overnight in 4% paraformaldehyde or Feketes fixative (29Shillingford J.M. Miyoshi K. Robinson G.W. Bierie B. Cao Y. Karin M. Hennighausen L. J. Histochem. Cytochem. 2003; 51: 555-565Crossref PubMed Scopus (51) Google Scholar) and paraffin embedded. Sections were deparaffinized in xylene, passed through a graded alcohol series and then either microwaved for 10 min, boiled at 97.5 °C for 30 min in 10 mm sodium citrate buffer (pH 6.0) or trypsinized with Digest-All 2 (Zymed Laboratories Inc.). After rinsing in water, sections were incubated in 2.5% hydrogen peroxide in methanol to block endogenous peroxidase activity. Sections were washed in PBS and blocked in 5% serum in PBS. Sections were incubated with primary antibody in 0.1% bovine serum albumin for 1 h at room temperature or overnight at 4 °C. After washing in PBS, sections used for immunohistochemistry were incubated with biotinylated secondary antibody (Vector Laboratories) in PBS containing 5% serum, washed in PBS, and incubated with streptavidin peroxidase (Zymed Laboratories Inc.). The sections were washed in PBS, visualized using the DAB substrate kit (Zymed Laboratories Inc.), and counterstained with hematoxylin. Alexa Fluor 488-conjugated goat anti-rabbit IgG and Alexa Fluor 546-conjugated goat anti-mouse IgG (all 1:300, Molecular Probes) were used for immunofluorescence labeling and the nuclei were stained using 5 μm Draq5 (Alexis Biochemicals). Primary antibodies: rabbit polyclonal anti-GLI1 (Abcam), 1:2000; rabbit polyclonal anti-Ptch1 generated using the C-terminal 18 amino acids as antigen (custom made by Research Genetics) 1:200; rabbit polyclonal anti-AQP5 (Alpha Diagnostics) 1:200; rabbit polyclonal anti-Snail (Abcam) 1:800; anti-Laminin (Sigma) 1:100; and rabbit polyclonal anti-Cyclin D1 (Labvision) 1:100. The rabbit polyclonal antibodies recognizing Slc12a2 1:1000 and Slc34a2 1:250 were obtained from Dr. Jim Turner (NIDCR, National Institutes of Health, Bethesda, MD) and Dr. Juerg Biber (University of Zurich, Switzerland), respectively. Detection of BrdUrd (1:25, BD Biosciences), E-cadherin (1:11000, BD Transduction Laboratories), smooth muscle actin (1:50, Novocastra), β-catenin (1:5000, BD Transduction Laboratories), and F4/80 (1:500, Serotec) was performed using mouse monoclonal antibodies and the Histomouse kit (Zymed Laboratories Inc.), according to the manufacturer's instructions. As negative controls, all experiments were performed without the primary antibody in the presence of equal concentrations of normal rabbit, goat, or mouse IgG. Northern Blot Hybridization—RNA from mammary gland tissues was obtained and the Northern blot was performed according to the NorthernMax™ (Ambion) manual. Probes against WAP, α-lactalbumin, β-casein, and Gapdh were a kind gift from Dr. Stephan Teglund (30Teglund S. McKay C. Schuetz E. van Deursen J.M. Stravopodis D. Wang D. Brown M. Bodner S. Grosveld G. Ihle J.N. Cell. 1998; 93: 841-850Abstract Full Text Full Text PDF PubMed Scopus (1066) Google Scholar). All probes were labeled with 32P using the High Prime labeling kit (Roche). Immunoprecipitation and Western Blot Analysis—Mammary fat pads were dissected, quickly frozen in liquid nitrogen, and homogenized in 10 mm Tris-HCl (pH 7.6), 5 mm EDTA, 50 mm NaCl, 30 mm sodium pyrophosphate, 50 mm sodium fluoride, 1 mm sodium orthovanadate, 10% glycerol with Complete protease inhibitor mixture (Roche Applied Science). Western blot analysis were performed on tissue protein lysates using the primary antibodies anti-GLI1 (1:4,000, Abcam), anti-E-cadherin (1:2,500, BD Biosciences), anti-STAT5 (1:1,000, Santa Cruz Biotechnologies), or anti-Actin (1:10,000, Sigma) and immunoprecipitated proteins using the primary antibody anti-P-STAT5 (Y694, 1:100 Epitomics) essentially as described else-where (31Agren M. Kogerman P. Kleman M.I. Wessling M. Toftgard R. Gene (Amst.). 2004; 330: 101-114Crossref PubMed Scopus (142) Google Scholar). Generation of Mice with Targeted Expression of the Hh Effector GLI1 in the Mammary Gland—To study the influence of a deregulated Hh signaling pathway during postnatal development in the mouse mammary gland, we employed a bigenic transgenic system to express the hedgehog effector GLI1 in the mammary epithelium (Fig. 1A). The reverse tet-responsive transactivator (rtTA) was expressed under the control of a mammary tumor virus long terminal repeat (MMTV LTR) and administration of doxycycline resulted in activation of the TRE-linked transgene, GLI1. The bitransgenic and WT animals were induced either continuously from mating or starting 3 weeks after birth. The time of GLI1 expression did not influence the phenotypes observed in the GLI1-induced transgenic mammary glands nor did it alter the GLI1 expression levels in the mammary gland during pregnancy as measured by RT-PCR (data not shown). The bigenic doxycycline-treated mice were normal from birth and indistinguishable from their WT littermates in weight, size, development, fertility, and behavior. All mice were genotyped by PCR analysis, confirming the presence of both the TREGLI1 (Fig. 1B, lanes 1 and 3-6) and the MMTVrtTA allele (Fig. 1B, lanes 1-5, 7, 8, and 10) in the bigenic mice (Fig. 1B, lanes 1 and 3-5). Expression of Hedgehog Pathway Components in WT and GLI1-expressing Mammary Glands—To investigate the expression pattern of Gli1 and Ptch1 in the mammary gland, RT-PCR and immunohistochemistry were performed at various developmental stages. The Gli1 primer pair used for RT-PCR was designed to recognize both human and mouse Gli1 mRNA. The RT-PCR analysis demonstrated that the expression of Gli1 and Ptch1 in the mammary gland from doxycycline-treated bigenic mice was enhanced during pregnancy at all time points analyzed (6.5 dpc, 18.5 dpc, and L1), when compared with WT mice (Fig. 1C). In addition, immunohistochemistry revealed enhanced cytosolic and nuclear expression of the GLI1 protein in mammary tissue from GLI1 expressing mice during pregnancy (6.5 and 18.5 dpc) and parturition (L1) when compared with WT mice at the same developmental stage (Fig. 1D, a-f). The GLI1 expression was essentially homogenous in the mammary epithelial cell compartment. However, a small fraction of predominantly luminal cells were GLI1 negative. The Ptch1 expression in the WT and induced bigenic mammary gland followed the GLI1 expression as expected due to the autoregulatory properties of the Hh signaling pathway (Fig. 1D, g-l) (32Hahn H. Wicking C. Zaphiropoulous P.G. Gailani M.R. Shanley S. Chidambaram A. Vorechovsky I. Holmberg E. Unden A.B. Gillies S. Negus K. Smyth I. Pressman C. Leffell D.J. Gerrard B. Goldstein A.M. Dean M. Toftgard R. Chenevix-Trench G. Wainwright B. Bale A.E. Cell. 1996; 85: 841-851Abstract Full Text Full Text PDF PubMed Scopus (1660) Google Scholar). The GLI1 and Ptch1 expression levels were below detection level in induced bigenic virgin (5 and 10 weeks) mice (data not shown). GLI1-expressing Mice Fail to Lactate—GLI1 transgenic females exposed to doxycycline gave birth to live pups of normal size and number with the expected Mendelian distribution of genotypes. Maternal behavior appeared normal with respect to nursing and the pups showed no craniofacial alterations or apparent neurological defects. All pups, irrespective of genotype, born to GLI1 expressing females died from failure to nurse and consequently no milk spots could be detected (Fig. 1E). The phenotype showed a 100% penetration rate among the GLI1 expressing mothers (n > 30) and could not be compensated for by subsequent pregnancies (n = 10). Furthermore, addition of doxycycline during the second or later pregnancies to previously un-induced females replicated these findings. Non-induced bigenic mice were able to nurse their pups and showed normal development of the mammary glands (data not shown). Expression of GLI1 Impairs the Lobuloalveolar Development of the Mammary Gland—Whole mount and immunohistological analysis were performed at different developmental stages and compared with WT mice. At 5 and 10 weeks of age, analysis of mammary glands from doxycycline-induced bigenic mice showed no alterations when compared with WT animals at the same age and the mammary fat pads were to an equal degree occupied with mammary epithelium (data not shown). In contrast, whole mounts from 6.5 dpc (Fig. 2A, a), 18.5 dpc (Fig. 2A, c), and L1 (Fig. 2A, e) demonstrated that induced bigenic mice had a reduced alveolar network, with minimal side branching and ductal sprouting, when compared with WT mice (6.5 dpc (Fig. 2A, b), 18.5 dpc (Fig. 2A, d), and L1 (Fig. 2A, f)). The number of secretory alveoli was reduced during pregnancy at all time points when compared with WT glands and the bigenic GLI1 expressing alveoli were smaller, with small or closed lumina (Fig. 2A, g-l). Variability in number and size of alveoli in the mammary glands of different bitransgenic females were observed, however, when compared with WT the bitransgenic alveoli were always fewer and less developed. Histological analysis confir" @default.
• W2087028407 created "2016-06-24" @default.
• W2087028407 creator A5032930701 @default.
• W2087028407 creator A5040572906 @default.
• W2087028407 creator A5042257111 @default.
• W2087028407 creator A5071903223 @default.
• W2087028407 creator A5086792761 @default.
• W2087028407 date "2007-12-01" @default.
• W2087028407 modified "2023-10-16" @default.
• W2087028407 title "Targeted Expression of GLI1 in the Mammary Gland Disrupts Pregnancy-induced Maturation and Causes Lactation Failure" @default.
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