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Altered mammary epithelial development, pattern formation and involution in transgenic mice expressing the EphB4 receptor tyrosine kinase

Nadia Munarini^1,*, Richard Jäger^2,*, Susanne Abderhalden¹, Gisela Zuercher¹, Valeria Rohrbach¹, Saemi Loercher¹, Brigitte Pfanner-Meyer¹, Anne-Catherine Andres¹ and Andrew Ziemiecki^1,

¹ Department of Clinical Research, University of Berne, Tiefenaustrasse 120, CH-3004 Bern, Switzerland
² Institute for Genetics, Karlsruhe Research Center, D-76021 Karlsruhe, Germany*Both authors contributed equally to this work

View larger version (50K): [in a new window]   Fig. 1. EphB4 transgene expression. (A) Northern blot analysis of transgene expression throughout mammary gland development. 10 µg of total RNA was prepared from mammary glands of control (C) and transgenic mice (T) at the developmental stages indicated and analysed using an EphB4-specific probe. (B) RT-PCR analysis of EphB4 transgene and ephrin-B2 expression during mammary gland development. RNA was prepared from mammary glands of control (C) and transgenic mice (T) at the developmental stages indicated and amplified using transgene and ephrin-B2-specific primers. Std, Molecular weight marker; +co, positive control using transgene DNA as template; –co, negative control without RNA. (C) Western blot analysis of EphB4 transgene expression. Protein extracts from mammary glands of control (1), heterozygous (2) and homozygous transgenic mice (3) at the developmental stages indicated were analysed using a murine EphB4-specific polyclonal antibody. (D) Tyrosine phosphorylation of the transgenic EphB4 protein. Protein extracts of lactating control and transgenic mammary glands were immunoprecipitated (IP) with either EphB4 (Eph) or phosphotyrosine (p-tyr) antibodies, the immunoprecipitates resolved by SDS-PAGE and western blotted with EphB4 antibodies. (E and F) Immunohistochemical localization of the EphB4 transgene protein. Sections of lactating mammary glands were reacted with affinity-purified EphB4 antibodies and visualized with peroxidase-coupled anti-rabbit IgG. Sections were counterstained with hematoxilin. Bars represent 25 µm.

View larger version (69K): [in a new window]   Fig. 2. Whole-mount staining of mammary glands of control and transgenic mice (transgene) at puberty (5.5 weeks) (A-D), in virgins at 10 weeks (E-H) and 13.5 weeks of age (I-L). Bars represent 1.5 cm (A,B,E,F,I,J) or 100 µm (C,D,G,H,K,L)

View larger version (102K): [in a new window]   Fig. 3. Cell proliferation in pubertal and virgin mammary glands. Mammary glands were prepared from control and transgenic (transgene) mice injected with BdUr at four weeks (puberty) and 10 weeks (virgin) of age. Sections were reacted with an anti-BdUr antibody visualized with peroxidase-coupled anti-mouse IgG. Counterstaining was with hematoxilin. Bars represent 50 µm.

View larger version (130K): [in a new window]   Fig. 4. Whole-mount staining and cell proliferation of mammary glands during pregnancy. Whole-mount staining (A-D) and BdUr detection (E,F) was performed on control and transgenic (transgene) mammary glands at day 18 of pregnancy. For BdUr detection, sections were reacted with a BdUr antibody visualized with peroxidase-coupled anti-mouse IgG and counterstained with hematoxilin. Bars represent 200 µm (A,B) or 50 µ (C-F).

View larger version (88K): [in a new window]   Fig. 5. Apoptotic cell death in mammary glands of control and transgenic (transgene) mice at 16 days (A,B) and 18 days (C,D) of pregnancy. Sections were subjected to the TUNEL assay using TMR red dUTP. Bars represent 50 µm (A,C) or 25 µm (B,D).

View larger version (86K): [in a new window]   Fig. 6. Histology of lactating mammary glands. Sections of lactating mammary glands from control and transgenic (transgene) animals at day 10 of lactation were stained with hematoxilin and eosin. Bars represent 25 µm.

View larger version (89K): [in a new window]   Fig. 7. Apoptotic cell death in mammary glands during involution. Sections of mammary glands from control and transgenic (transgene) mice at the time points of involution indicated were subjected to the TUNEL assay using TMR red dUTP. Bars represent 25 µm (A,B,C,D,H) or 50 µm (E,G,F).

View larger version (90K): [in a new window]   Fig. 8. Cell proliferation in mammary glands at involution. Sections of mammary glands from BdUr-injected control (A) and transgenic (B; transgene) mice at day two of involution were reacted with an anti-BdUr antibody visualized with peroxidase-coupled anti-mouse IgG. Sections were counterstained with hematoxilin. Bars represent 25 µm. (C) Clusterin expression during mammary gland involution. Northern blot analysis of clusterin expression. 10 µg of total RNA prepared from mammary glands of control (C) and transgenic (T) animals at the developmental stages indicated were analysed. (D) Western blot analysis of EphB4 expression. Protein extracts prepared from mammary glands of control (C) and transgenic (T) animals at different time points of involution (invol.)

View larger version (110K): [in a new window]   Fig. 9. Histology of involuting mammary glands. (A-F) Sections of mammary glands of control and transgenic (transgene) animals taken at the indicated time points after weaning were stained with hematoxilin and eosin. Bars represent 50 µm. (G,H) Whole-mount staining of regressed mammary glands from control (G) and transgenic (H; transgene) animals taken 4.5 weeks after weaning. Bars represent 200 µm.

View larger version (60K): [in a new window]   Fig. 10. Mammary tumour formation in NeuT and NeuT/EphB4 double transgenic mice (A) Table summarizing tumour frequency, latency and metastasis formation. (B-D) Histology of primary mammary tumours from NeuT transgenic mice (B), of primary mammary tumours (C) and lung metatstasis (D) from EphB4/NeuT double transgenic mice. Sections were stained with hematoxilin and eosin. Bars represent 50 µm.