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First published online 8 December 2004
doi: 10.1242/dev.01583

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Targeted activation of ß-catenin signaling in basal mammary epithelial cells affects mammary development and leads to hyperplasia

¹ UMR 144 CNRS-Institut Curie, Institut Curie, Section de Recherche, 26 rue d'Ulm, 75248, Paris, Cedex 05, France
² Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot, 76100, Israel

View larger version (34K): [in a new window]   Fig. 1. Transgene and its expression in the mouse mammary gland. (A) The ß-catenin construct used for expression under the control of the K5 promoter in the basal mammary epithelial cells. (B) RT-PCR analysis of transgene expression in 7.5-day-pregnant mouse mammary glands. 18S RNA was used as a normalization control for RT-PCR. (C) Western blotting analysis of 7.5-day-pregnant wild-type and transgenic mouse mammary gland protein extracts performed with anti-HA-tag antibody. ß-Actin served as loading control. (D) Double immunofluorescence labeling showing transgene expression in a mammary duct of K5-N57-ßcat mouse. The transgene product detected with anti-HA-tag antibody is found in the basal cell layer of the mammary epithelium in correlation with the K5 expression, whereas the luminal epithelial cell layer is not labeled (arrows). DAPI was used to stain the nuclei. L, lumen. Scale bar: 10 µm.

View larger version (60K): [in a new window]   Fig. 2. Expression of N57-ß-catenin in basal mammary cells induces precocious development in pregnancy, persistent proliferation in lactation and accelerated involution. (A) Precocious lobuloalveolar development in pregnant K5-N57-ßcat mice. Wholemount staining of mammary glands from pregnant wild-type (WT) and transgenic mice. (B) Premature secretory cell differentiation in pregnant K5-N57-ßcat mice. ß-Casein expression in 7.5-day-pregnant mouse mammary glands. Alveoli from K5-N57-ßcat mice contain more ß-casein than those from wild-type littermates. Immunostaining with anti-ß-casein (green) and anti--SM-actin (red) antibodies. (C) Persistent proliferation in K5-N57-ßcat mouse mammary glands at peak lactation. Staining with anti-BrdU antibodies (upper panel) and TUNEL assay (lower panel). The number of BrdU-positive cells (arrows) is greater in lactating transgenic mouse mammary glands after 6 days of lactation than in wild-type animals, whereas apoptosis rates are not altered. (D) Accelerated involution in K5-N57-ßcat mammary glands as revealed by Hematoxylin and Eosin staining (HE, top panel) and TUNEL assay (bottom panel). Sections through 3-day-old involuting mammary glands. P7.5, P10.5 and P13.5 correspond to 7.5, 10.5 and 13.5 days of pregnancy; L6, to 6 days of lactation; I3, to 3 days of involution. Scale bars: 4 and 0.5 mm in A; 20 µm in B; 100 µm in C; 200 µm in D (upper panel), 100 µm in D (lower panel).

View larger version (30K): [in a new window]   Fig. 3. Milk gene expression in wild-type (WT) and K5-N57-ßcat mammary glands estimated by semi-quantitative RT-PCR at different developmental stages. There are increased levels of ß-casein in 7.5-day-pregnant, increased levels of WAP and -lactalbumin (-LA) in 13.5-day-pregnant, and an accelerated decrease of ß-casein, WAP and -LA in involuting K5-N57-ßcat mouse mammary glands. 18S RNA was used as a normalization control for RT-PCR. P7.5, P10.5 and P13.5 correspond to 7.5, 10.5 and 13.5 days of pregnancy; L2 and L6, to 2 and 6 days of lactation; I3, to 3 days of involution.

View larger version (48K): [in a new window]   Fig. 4. Altered expression of matrix-degrading metalloproteinases and their inhibitors in K5-N57-ßcat mouse mammary glands. Semi-quantitative RT-PCR analysis of Mmp and Timp transcript levels in wild-type (WT) and K5-N57-ßcat (K5-Nß) mouse mammary glands. 18S RNA was used as a normalization control for RT-PCR. Samples from wild-type and K5-N57-ßcat glands were analyzed in the same gel. P7.5, P10.5 and P13.5 correspond to 7.5, 10.5 and 13.5 days of pregnancy; L2 and L6, to 2 and 6 days of lactation; I3, to 3 days of involution.

View larger version (17K): [in a new window]   Fig. 5. Real-time RT-PCR analysis of Myc, cyclin D1 and p63 transcript levels in 7.5-day-pregnant mouse mammary glands. Myc, cyclin D1, TAp63 and Np63 transcripts were quantified as described in the Materials and methods. Bars represent the mean values±s.e.m. for five wild-type (WT) and five K5-N57-ßcat (K5-Nß) mice normalized to the Gapd mRNA levels. The difference in the Myc, cyclin D1 and Np63 transcript levels between transgenic and wild-type mice was statistically significant (P<0.0001, 0.001 and 0.02, respectively).

View larger version (112K): [in a new window]   Fig. 6. Basal-type mammary hyperplasia in 12- to 18-month-old K5-N57-ßcat mice. (A) Whole-mount staining of 18-month-old wild-type (WT) and K5-N57-ßcat nulliparous mouse mammary gland fragments containing large ducts. (B) Sections through two 18-month-old K5-N57-ßcat nulliparous mouse mammary glands stained with anti-K5 antibody. Note focal thickenings of ductal epithelium (arrows, upper panel) and large hyperplastic areas (lower panel) containing K5-positive cells. (C) Double indirect immunofluorescence staining of a section through a 16-month-old nulliparous. K5-N57-ßcat mouse mammary gland containing focal epithelial thickenings with anti-K5 and anti-HA-tag-antibodies revealing transgene product. There are numerous HA-positive nuclei (arrows). (D,E) Serial sections through a wild-type (WT) and a 16-month-old K5-N57-ßcat nulliparous mouse mammary gland containing focal epithelial thickenings double stained with anti-K5 and anti-K8 (D) or with anti-K5 and anti--SM-actin-antibodies (E). Cells in the hyperplastic areas are K5 positive, but do not express K8 or -SM-actin. The broken line indicates the position of the basement membrane. (F) Double indirect immunofluorescence staining of a section through a hyperplastic area from a 12-month-old multiparous K5-N57-ßcat mouse mammary gland with anti-laminin and anti-p63 antibodies. The staining of the basement membrane with the anti-laminin antibody is discontinuous around hyperplastic ducts filled with p63-expressing cells (arrows). Scale bars: 0.6 mm in A; 0.2 mm in B; 30 µm in C-E; 50 µm in F.

View larger version (155K): [in a new window]   Fig. 7. Mammary lesions in multiparous K5-N57-ßcat mice. (A-C) Squamous metaplasia in a 12-month-old K5-N57-ßcat mouse mammary gland. Double immunolabeling with anti-K5 and anti-K10 antibodies (A), anti-K5 and anti hair keratin (B), or immunostaining with anti-ß-catenin antibody (C). ß-catenin-positive nuclei are present (C, arrows). (D) Section through an invasive mammary carcinoma from a 12-month-old K5-N57-ßcat mouse stained with anti-K5 and anti-K8 antibodies. Double indirect immunofluorescence. (E) Section through the same tumor stained with anti-ß-catenin and anti-cyclin D1 antibodies. Numerous nuclei stain positive with both antibodies. Scale bars: 50 µm in A,B,D,E; 30 µm in C.

View larger version (18K): [in a new window]   Fig. 8. Levels of Myc, cyclin D1, Timp3 and p63 transcripts in the mammary hyperplasia in K5-N57-ßcat mice. (A) Real-time RT-PCR analysis of levels of Myc, cyclin D1 and Timp3 transcripts in hyperplastic and tumor-free tissue samples from 12- to 18-month-old transgenic glands (K5-Nß). Bars represent the mean values x10-2±s.e.m. obtained in two independent experiments performed in duplicate. (B) Real-time RT-PCR analysis of TAp63 and Np63 transcript levels in wild-type and transgenic glands. Bars represent the mean values x10-4±s.e.m. obtained for RNA samples isolated from hyperplastic and tumor-free areas from four 12- to 18-month-old K5-N57-ßcat (K5-Nß) and four wild-type (WT) mice. The differences in Np63 transcript levels were statistically significant between wild-type and transgenic tumor-free specimens, wild-type and transgenic hyperplastic tissue, and transgenic tumor-free and transgenic hyperplastic tissue (P<0.003, 0.001 and 0.002, respectively). The differences in TAp63 transcript levels were statistically significant between wild-type and transgenic tumor-free specimens, and between wild-type and transgenic hyperplastic tissue (P<0.0015 and 0.002, respectively). Transcript levels were normalized to the Gapd mRNA.