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First published online 1 September 2004
doi: 10.1242/dev.01347

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Canonical WNT signaling promotes mammary placode development and is essential for initiation of mammary gland morphogenesis

Emily Y. Chu^1,2,*, Julie Hens^3,*, Thomas Andl¹, Alladin Kairo¹, Terry P. Yamaguchi⁴, Cathrin Brisken⁵, Adam Glick⁶, John J. Wysolmerski^3, and Sarah E. Millar^1,2,,

¹ Departments of Dermatology and Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
² Cell and Molecular Biology Graduate Group, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA
³ Section of Endocrinology and Metabolism, Department of Internal Medicine, Yale University, New Haven, CT 06520, USA
⁴ Cancer and Developmental Biology Laboratory, National Cancer Institute-Frederick, Frederick, MD 21702, USA
⁵ Swiss Institute for Experimental Cancer Research, NCCR Molecular Oncology, CH-1066 Epalinges s/Lausanne, Switzerland
⁶ Laboratory of Cellular Carcinogenesis and Tumor Promotion, National Cancer Institute, Bethesda, MD 20892, USA

View larger version (85K): [in a new window]   Fig. 1. TOPGAL expression during mammary gland development. (A) Development of mammary buds (red circles) from the presumptive mammary line (broken red line) during mouse embryogenesis (E10.5-E12.5). Mammary buds 1 and 5 develop underneath the limb buds and are depicted as open circles with a broken circumference. Placodes arise asynchronously, with placode pair number 3 detected first, followed by number 4, then numbers 1 and 5, and finally pair number 2 (Veltmaat et al., 2003). (B-G) Whole-mount X-gal stained TOPGAL embryos at E10.5 (B,C); E11.5 (D-F); and E12.5 (G). Cells expressing the TOPGAL transgene appear blue. At E10.5, TOPGAL activity appears as a streak in the mammary region between the fore- and hind-limb buds (lb), indicated by arrows (B,C). By E11.5, TOPGAL-positive cells accumulate in placodes at defined locations that are reproducible between different embryos (D-F). D' shows a higher magnification view of placode number 3 from the embryo shown in D. Blue-stained cells are seen on the surface of the embryo adjacent to the developing placode (arrows). At E12.5, almost all the blue-staining cells in the mammary region are concentrated within well-defined mammary buds (G, arrows). (H) X-gal-stained whole-mount of E17.5 ventral skin showing punctate staining throughout the mammary epithelial ducts. (I) 20 µm section through the area of the mammary line in an E10.5 TOPGAL embryo. Note TOPGAL activity in both the surface epithelium and the underlying mesenchyme (arrow). (J) 5 µm section through a mammary bud from an E13.5 embryo. The epithelial cells and a few surrounding mammary mesenchymal cells (arrow) express TOPGAL. (K) 5 µm section through the mammary ducts from an E17.5 embryo. A subset of epithelial cells and a few mesenchymal cells express TOPGAL (arrows). (L) Whole-mount preparation of X-gal and carmine aluminum (red)-stained mammary gland from a TOPGAL transgenic mouse on day 13 of pregnancy. TOPGAL is active in developing alveoli (arrow). (M) 5 µm section through the mammary gland shown in L. TOPGAL is active in epithelial cells of developing alveolar structures (arrows).

View larger version (104K): [in a new window]   Fig. 2. Expression of WNT pathway genes at early stages of mammary development. (A,B) In-situ hybridization of a 35S-labeled probe for Wnt3 to a parasagittal paraffin section of an E10.5 embryo photographed in bright field (A) and dark field (B) with red stage illumination. The signal appears as red grains and is indicated by arrows. (C-L) Whole-mount in-situ hybridization using the probes indicated. The signal appears red-brown and mammary region expression is indicated by arrows in each panel. Insets in panels C,E,F,G represent higher magnification photographs of the regions indicated by arrows in each panel.

View larger version (92K): [in a new window]   Fig. 6. Absence of localized expression of mammary placode markers in induced K5-rtTA, tetO-Dkk1 embryos and presence of mammary rudiments in Wnt5a-null embryos. (A-P) K5-rtTA, tetO-Dkk1 double transgenic (DT) (B,D,F,H,J,L,N,P) and single transgenic (ST) control littermate (A,C,E,G,I,K,M,O) embryos that were doxycyline-treated throughout gestation, harvested at E12.5 (A-J), E11.75 (K,L), E11.5 (M,N) and E10.5 (O,P), and subjected to whole-mount in-situ hybridization with the probes indicated. Control E12.5 and E11.75 embryos show discrete upregulation of ß-catenin (A), Wnt10b (C), Krm2 (I) and Tbx3 (K) in mammary buds (A,C,I) and placodes (K). Limbs were removed from the embryo in A to show all five buds on the left side. Localized expression of all of these genes is absent from the mammary region of induced K5-rtTA, tetO-Dkk1 double transgenic littermates (B,D,J,L). Wnt11 is weakly expressed in ring shapes around the buds of control (G) (blue arrows) but not Dkk1-expressing (H) embryos. Weak expression of Wnt11 in a broad stripe of mesenchyme is present in both control and Dkk1-expressing embryos (G,H, yellow arrows). Expression of Wnt5a in mammary region mesenchyme is unaffected by ectopic Dkk1 (E,F) (arrows). Upregulated Wnt10b expression is first apparent in the mammary line of controls at E11.5 (M, arrow), and is absent in the induced K5-rtTA, tetO-Dkk1 double transgenic littermate (N). Fgf10 is expressed in somites of doxycyline-treated control and K5-rtTA, tetO-Dkk1 double transgenic littermates at E10.5 (O,P, arrows). (Q,R) Transverse hematoxylin and eosin-stained paraffin sections of E15.5 Wnt5a+/– control (Q) and Wnt5a–/– (R) female embryos showing thoracic mammary rudiments (arrows). (S,T) Wnt10b expression in mammary buds of Wnt5a+/– (S) and Wnt5a–/– (T) E12.5 embryos (arrows).

View larger version (102K): [in a new window]   Fig. 3. Mammary placode formation in cultured embryos. (A-C) X-gal-stained E10.5 TOPGAL embryos after 0 hours (A), 24 hours (B) or 48 hours (C) culture in control medium. Arrows indicate developing placodes. (D-F) X-gal-stained E11 TOPGAL embryos after 24 hours culture in control-conditioned medium (D), WNT3A-conditioned medium (E) or LiCl-supplemented medium (F). WNT3A treatment causes the formation of placodes that are larger and more distinct than in the littermate control (arrows in D,E). LiCl treatment causes activation of TOPGAL transgene expression along the lateral aspect of the embryo, and formation of multiple intensely blue-staining clusters of cells (F, arrows). (G-I) Histological sections (counterstained with eosin) through the mammary placodes of E11 X-gal-stained embryos cultured for 24 hours in control medium (G), WNT3A-conditioned medium (H) or LiCl (I). (J-K) Whole mount in-situ hybridizations for Wnt10b on E11 embryos cultured for 48 hours (J,K) or 24 hours (L) in control medium (J), WNT3A-conditioned medium (K) or 50 mM LiCl (L). LiCl treatment was limited to 24 hours, as we found 48 hours' exposure to LiCl to be toxic. Arrows indicate Wnt10b expression in placodes (J-L) and ectopic placode-like structures (L). Scale bar: 0.65 mm in A; 0.75 mm in B; 0.9 mm in C; 0.5 mm in D-F, 33 µm in G-I; 0.6 mm in J-L.

View larger version (65K): [in a new window]   Fig. 4. Inducible expression of Dkk1 in the surface epithelium and inhibition of TOPGAL activity. (A) Scheme for doxycycline-inducible expression of Dkk1. Mice carrying the K5-rtTA transgene (yellow) are mated to mice carrying the tetO-Dkk1 transgene (blue). In double transgenic offspring (green), expression of Dkk1 can be induced by doxycycline in cells where the K5 promoter is active. (B) Phenotype of newborn double transgenic (DT) pup (right) compared with control single transgenic (ST) littermate (left) after doxycycline treatment from E0.5. The DT pup has severe limb defects (arrows), open eyes and lacks vibrissa follicles. (C-F) A K5-rtTA, tetO-Dkk1, TOPGAL (Dkk1-expressing/TOPGAL) embryo (D,F) and tetO-Dkk1, TOPGAL (Control/TOPGAL) control littermate (C,E) were doxycycline-treated throughout gestation, harvested at E11.5 and stained with X-gal to reveal sites of TOPGAL WNT reporter expression. (E,F) Higher-magnification photographs of the mammary regions of the embryos shown in C,D, respectively. Strong staining for ß-galactosidase is visible in the mammary region of the control at sites of mammary placode development (C,E, red arrows) and other sites, including the apical ectodermal ridge of each limb bud. TOPGAL activity was severely reduced in the mammary region of the K5-rtTA, tetO-Dkk1, TOPGAL embryo (D,F). Deformities of the limb buds correlate with loss of ß-galactosidase expression from the limb edge (yellow arrows in D), consistent with the known requirement for canonical WNT signaling in maintenance of the apical ectodermal ridge (Barrow et al., 2003).

View larger version (166K): [in a new window]   Fig. 5. Mammary placodes are absent in Dkk1-expressing embryos. Scanning electron microscopy of the ventral-lateral surface of induced K5-rtTA, tetO-Dkk1 (B,D) and control single transgenic littermate (A,C) embryos at E11.5. Placode number 3 is visible in the control as a mound of cells protruding above the surface of the embryo (arrows in A,C) and is absent in the induced double-transgenic embryos (B,D). A,B were photographed at 75x magnification and C,D at 200x magnification. Scale bars: 100 µm. lb, limb bud.

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