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First published online 7 February 2007
doi: 10.1242/dev.02797

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Constitutive activation of smoothened (SMO) in mammary glands of transgenic mice leads to increased proliferation, altered differentiation and ductal dysplasia

Baylor Breast Center and Department of Molecular and Cellular Biology, Room N1210; MS:BCM600, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

View larger version (69K): [in this window] [in a new window]   Fig. 1. Whole-mount and histological analysis of mammary glands from wild-type and MMTV-SmoM2 mice. (A) Whole-mount showing a normal TEB array from a wild-type mouse at 5 weeks of age. Terminal structures are indicated with an asterisk; side-buds are arrowed. (B) Whole-mount showing a TEB array from an MMTV-SmoM2 transgenic mouse at 5 weeks of age. Symbols are as described in A. (C) Quantification of TEB number in wild-type versus MMTV-SmoM2 mice at 5 weeks of age. (D) Histological preparation of a TEB in a wild-type gland showing organized cap and body cell layers, as well as normal periductal stroma condensing around the neck of the TEB (arrow). (E) Histological preparation of TEB in an MMTV-SmoM2 transgenic gland showing abnormal histology associated with the side-budding phenotype (arrow). TEBs can also show disorganized cap and body cell layers (inset). (F) Whole-mount from a 10-week-old wild-type mouse showing normal duct patterning and blunt-ended ducts (arrowhead). (G) Whole-mount from a 10-week-old MMTV-SmoM2 mouse showing an increase in side budding and branching (arrowhead). Inset shows retained TEB-like structures. (H) Branch-point analysis and quantification of retained TEB-like structures at 10 weeks of age as a function of genotype. (I) Histological preparation of a normal mature duct in a wild-type gland. (J) Histological preparation of a mature duct in an MMTV-SmoM2 gland. Inset shows retained TEB-like structures (arrows). (K,L) Dual immunofluorescence for SMO and SMA in (K) wild-type and (L) MMTV-SmoM2 ducts. Arrowheads indicate SMO-expressing cells. Scale bars: 50 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 2. Dual immunofluorescence deconvolution microscopy for SMO and PTCH1 in wild-type and MMTV-SmoM2 transgenic mammary glands. Expression of SMO (A,D), PTCH1 (B,E) and merged three-color images including the DAPI counterstain (C,F) for wild-type (A-C) and MMTV-SmoM2 transgenic (D-F) mammary glands. (A) No detectable SMO expression. (B) Near uniform PTCH1 expression. (C) Merge of A and B. (D) Area of duct in a transgenic gland showing ectopic SMO expression. (E) PTCH1 expression in the same duct showing cell to cell variation in PTCH1 staining. (F) Merge of D and E showing (arrowed) SMO+ cells in which PTCH1 is moderately elevated relative to SMO- cells. Scale bar: 50 µm.

View larger version (114K): [in this window] [in a new window]   Fig. 3. Proliferation and caspase-3-mediated apoptosis as a function of genotype at 10 weeks of age. Each marker is shown at the top of the column to which it applies. Genotype of the mouse from which the gland was derived is shown to the left of the row to which it applies. Arrows highlight stained cells or regions of epithelium expressing the marker. (A) BrdU and Ki67 (inset) staining in wild type. (B) Cleaved caspase-3 staining showing rare apoptotic cells. (C) BrdU and Ki67 (inset) staining in an MMTV-SmoM2 gland. (D) Cleaved caspase-3 staining showing a rare apoptotic cell. (E,F) BrdU and cleaved caspase-3 staining, respectively, in a Ptch1/+ gland displaying the characteristic ductal phenotype.

View larger version (62K): [in this window] [in a new window]   Fig. 4. Dual immunofluorescence analysis of MMTV-SmoM2 mice at 10 weeks of age. (A) SMO-Ki67 showing no colocalization. (B) SMO-ER showing no colocalization. (C) ER-Ki67 showing low frequency colocalization, confirmed by BrdU-ER dual staining (inset, arrowhead). (D) ER-CK6 showing high frequency colocalization. (E) Duct showing ectopic CK6 expression (arrowheads), but no detectable SMO expression. (Inset) A region of high SMO expression lacking CK6 expression (arrowhead).

View larger version (34K): [in this window] [in a new window]   Fig. 5. Effect of MMTV-SmoM2 on mammosphere formation and regeneration of the mammary gland. (A) Pairwise comparison of log-transformed mammosphere-formation efficiency values for five paired sets of primary epithelial cell preparations. MMTV-SmoM2 cells showed a 2-fold increase in mammosphere-forming efficiency relative to wild-type cells in four of the five sample pairs. Key to symbols: open circles, MMTV-SmoM2; red filled circles, MMTV-SmoM2 average; open trianges, wild type; blue filled triangles, wild-type average. (B) Statistical evaluation of mammosphere-forming efficiency as a function of genotype, including all five paired samples shown in A. (C) Photomicrographs of representative mammospheres from wild-type and MMTV-SmoM2 mice. (D) Photomicrographs of representative outgrowths of transplanted mammospheres derived from wild-type and MMTV-SmoM2 mice. (E) Limiting-dilution transplantation analysis as a function of genotype. (F) Photomicrographs of representative outgrowths from limiting-dilution transplantations (100 cells) derived from wild-type and MMTV-SmoM2 mice, respectively. Inset shows a strongly affected outgrowth with increased side budding and branching.

View larger version (66K): [in this window] [in a new window]   Fig. 6. PTCH1 and SMO protein expression in human breast cancer. Representative expression patterns are shown with total immunohistochemical score for the sample, with the proportion and intensity scores in parentheses. PTCH1 is readily detectable in normal breast epithelium and isolated stromal cells (A), but shows a relative loss in the epithelium in DCIS (B) and IBC (C) (P<0.0001). SMO expression is undetectable in normal breast (D), but is detectable in the cytoplasm in 70% of DCIS (E) and 30% of IBC (F) (P<0.0001). (G) Average total IHC scores for SMO and PTCH1 in clinical samples of human breast.

View larger version (79K): [in this window] [in a new window]   Fig. 7. Dual immunofluorescence analysis of PTCH1-positive and SMO-positive human DCIS and IBC. (A-F) PTCH1-Ki67 in DCIS and IBC. Representative staining patterns are shown that indicate extensive colocalization of PTCH1 with Ki67 in those samples expressing detectable PTCH1. (G-J) SMO-Ki67 in DCIS and IBC. Representative staining patterns are shown that indicate rare colocalization of SMO with Ki67 in those samples expressing detectable SMO. Arrows and arrowheads in G indicate SMO+ cells and Ki67+ cells, respectively. (K) Quantitative analysis of SMO-Ki67 colocalization in DCIS and IBC.

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