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First published online 19 September 2007
doi: 10.1242/dev.006585

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The pattern of ß-catenin responsiveness within the mammary gland is regulated by progesterone receptor

¹ Department of Cell Biology, NYU School of Medicine, MSB 618, 550 1st Avenue, New York, NY 10016, USA.
² Department of Molecular and Cellular Biology, M533A, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
³ Department of Dermatology, NYU School of Medicine, MSB 618, 550 1st Avenue, New York, NY 10016, USA.

View larger version (113K): [in this window] [in a new window]   Fig. 1. N89ß-catenin expression rescues alveologenesis in PR-/- mice during pregnancy. Carmine and X-Gal-stained wholemounts of transplanted glands at 8.5 (A-D) and 14.5 (E-H) days of pregnancy. PR-/- glands do not develop alveoli (A,E). By contrast, alveoli are observed in PR-/-;N89ß-catenin glands (B,F) that are consistently more distended than in control PR+/- (C,G) and endogenous Rag1-/- glands (D,H). Scale bars: 200 µm.

View larger version (90K): [in this window] [in a new window]   Fig. 2. N89ß-catenin expression induces alveolar development. Immunohistochemistry demonstrating expression of casein in alveoli from endogenous Rag1-/- (A) and transplanted PR-/-;N89ß-catenin (B) glands at 14.5 days of pregnancy. Arrows point to luminal secretions containing casein. Immunohistochemical analysis demonstrating high levels of NKCC1 expression in young wild-type virgin (C) and transplanted PR-/- (D) glands at 14.5 days of pregnancy. Low levels of NKCC1 expression are observed in alveoli (arrowheads) generated in PR-/-;N89ß-catenin (E) and endogenous Rag1-/- (F) glands at p14.5. Insets show higher magnifications of the boxed areas and demonstrate NKCC1 expression along the basolateral cell borders of ductal cells (insets in C,D) and absence of NKCC1 staining in alveoli (insets in E,F). (G) An example of the criteria used to identify primary ducts, secondary branches and side-branches. (H) Table of the number of secondary and side-branches observed per 1000 pixels of ductal length in PR+/- (n=7 fields), PR+/-;N89ß-catenin (n=4 fields), PR-/- (n=7 fields) and PR-/-;N89ß-catenin glands (n=6 fields). Differences in side-branches are statistically significant between PR+/- and PR-/- glands (P=0.008) and PR+/-;N89ß-catenin and PR-/-;N89ß-catenin glands (P=0.003), but not between PR-/- and PR-/-;N89ß-catenin glands (P=0.057). Scale bars: 50 µm.

View larger version (130K): [in this window] [in a new window]   Fig. 3. Morphology of female virgin glands shows that N89ß-catenin induces precocious development in the presence and absence of PR. Carmine-stained wholemounts of inguinal mammary glands from 12-week-old (A-D) and 26-week-old (E-H) virgin mice. Precocious development is not observed in PR+/- (A,E) or PR-/- (C,G) glands but is seen in PR+/-;N89ß-catenin (B,F) and PR-/-;N89ß-catenin (D,H) glands, respectively. LN, lymph node. Brackets (F,H) indicate the regions that are magnified in Fig. 4. Scale bars: 5 mm.

View larger version (97K): [in this window] [in a new window]   Fig. 4. Absence of PR restricts N89ß-catenin-induced precocious development to ductal tips. Carmine-stained wholemounts show precocious development along the lateral borders and tips of ducts in PR+/-;N89ß-catenin glands (A) and terminally restricted precocious development in PR-/-;N89ß-catenin glands (B). Indirect immunofluorescence for the N-terminal Myc tag epitope detected by the 9E10 antibody shows that transgene expression in all luminal cells is unaffected by PR genotype (C,D). Scale bars: 0.3 mm in A,B; 100 µm in C,D.

View larger version (69K): [in this window] [in a new window]   Fig. 5. ß-catenin responsiveness is restricted to a subset of PR-negative mouse mammary cells, despite transgene expression in all cells. (A) Double immunofluorescence analysis of transgene detected by 9E10 (green) and PR expression detected by anti-PR (red) antibodies in a virgin MMTV-N89ß-catenin gland. (B) Expression of conductin-lacZ detected by X-Gal staining (blue, arrowheads) indicates that only a subset of ductal cells show a transcriptional response to uniform transgene expression immunohistochemically detected by 9E10 (brown) in a section of a PR+/+;conductin+/lacZ;N89ß-catenin gland. (C) PR immunohistochemistry on X-Gal-stained sections of PR+/+;conductin+/lacZ;N89ß-catenin glands demonstrates distinct populations of ß-catenin-responsive and PR-expressing cells. (D) Carmine and X-Gal-stained wholemount of a PR+/+;conductin+/lacZ;N89ß-catenin gland showing conductin-lacZ expression at sites of alveolar development. (E) Indirect immunofluorescence for detection of PCNA (green) and PR (red) reveals that proliferating cells are segregated from PR-expressing cells. (F) Immunohistochemistry for Ki67 on PR+/+;conductin+/lacZ;N89ß-catenin gland showing proliferation of one third of conductin-lacZ-expressing cells (arrows). Adjacent cells that do not express conductin-lacZ also proliferate. (G) Graphical representation of the percentage of Ki67-positive cells within PR-positive, conductin-lacZ-positive and conductin-lacZ-negative populations. Scale bars: 50 µm in A-C,E,F; 0.3 mm in D.

View larger version (61K): [in this window] [in a new window]   Fig. 6. A comparison of PR+/-;N89ß-catenin and PR-/-;N89ß-catenin glands. (A,C,E) PR+/-;N89ß-catenin; (B,D,F) PR-/-;N89ß-catenin. Hematoxylin and Eosin staining (A,B) shows that N89ß-catenin induces bilayered, secretion-filled structures. Note that alveolar lumen diameters are significantly larger in PR-/-;N89ß-catenin glands. Functional alveolar differentiation is demonstrated by immunohistochemistry for casein expression (C,D) and Oil Red O staining (E,F) to detect lipid droplets in alveoli. (G) Average alveolar luminal area in PR+/-;N89ß-catenin (gray) and PR-/-;N89ß-catenin (black) glands. Scale bars: 50 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 7. Northern blot analysis of milk protein RNA levels in 26-week-old virgin female mice. 28S and 18S rRNA subunits visualized by ethidium bromide staining provided loading controls. In addition, K18 (Krt18), a luminal cell marker, was used as a positive control for the integrity of the RNA and epithelial content. Three mice of each genotype were examined as indicated: PR+/- (lanes 2-4), PR+/-;N89ß-catenin (lanes 8-10), PR-/-;N89ß-catenin (lanes 11-13) and PR-/- (lanes 5-7). RNA from lactating (L, lane 14) and virgin (V, lane 1) mice were used as positive and negative controls, respectively.

View larger version (30K): [in this window] [in a new window]   Fig. 8. Schematic of the proposed interactions between PR and ß-catenin signaling. In PR-/- glands, cells at ductal tips are intrinsically responsive to ß-catenin signaling (blue). In PR+/- glands, PR induces competence to respond to ß-catenin signaling within a subset of alveolar progenitors along lateral borders (blue) and possibly designates other bipotent (gray) and ductal (yellow) progenitor populations. ß-catenin induces alveologenesis at ductal tips of PR-/- mice and at tips and along lateral borders of PR+/- mice. It also triggers the secretory differentiation program, resulting in distended secretion-filled lumen (pink) in PR-/- mice. Secretory differentiation is restrained by PR. In the normal virgin mammary gland, PR induces a non-uniform PR expression pattern and competence to respond to ß-catenin in alveolar progenitors (blue) during ductal maturation. During early pregnancy, PR-WNT4 signaling induces expansion of ductal progenitors (yellow) to form side-branches through ß-catenin-independent routes. PR and PRL cooperate to induce alveologenesis, a process that is ß-catenin-dependent. Later in pregnancy, PR restrains and PRL promotes alveolar differentiation. ß-catenin is required for alveologenesis and may participate at multiple steps in the secretory differentiation pathway.