An adjunct mammary epithelial cell population in parous females: its role in functional adaptation and tissue renewal
Kay-Uwe Wagner1,*,
Corinne A. Boulanger2,
MaLinda D. Henry1,
Magdalene Sgagias1,
Lothar Hennighausen3 and
Gilbert H. Smith2,*
1 Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, 986805 Nebraska Medical Center, Rm. 8009, Omaha, NE 68198-6805, USA
2 Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bldg. 10, Room 8B07, 9000 Rockville Pike, Bethesda, MD 20892-1750, USA
3 Laboratory of Genetics and Physiology, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bldg. 8, Rm. 107, Bethesda, MD 20892-0822, USA
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Fig. 1. Experimental design. The basic principle of the genetic labeling of differentiating cells in the developing mammary gland using the Cre-lox technique. In this experimental setting, the WAP-Cre transgene is used solely to monitor the differentiation process of alveolar precursor cells in response to lactogenic hormones. The transient upregulation of Cre recombinase in differentiating epithelial cells during pregnancy permanently activates a ubiquitously expressed reporter transgene (Rosa-lacZ), whose expression is not dependent on the differentiation status of a given cell. The reporter gene remains active in cells that no longer require high systemic hormone levels to maintain a functionally differentiated state (i.e. WAP gene expression). Hence, the permanent activation of the reporter gene (blue X-Gal staining) genetically labels differentiating cells that bypass apoptosis and remodeling at the conclusion of the reproductive cycle. The labeled cells in the remodeled (involuted) gland represent a new epithelial subtype, which is not present in nulliparous animals.
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Fig. 2. X-Gal stained mammary whole mounts and their histological sections of WAP-Cre/Rosa-lacZ double transgenic females (A-C,G-J) and their single transgenic controls (D-F). (A,D) Whole mount, first pregnancy, day 14 of gestation. (B,E) Whole mount, several hours post partum after the first pregnancy. (C,F) Whole mount, 3 weeks after weaning of the litter following the first gestation cycle. (G,I) Tissue sections from nulliparous (virgin) double transgenic mice counterstained with nuclear Fast Red. (H,J) Tissue sections from parous, non-pregnant (i.e. involuted) double transgenic females counterstained with nuclear Fast Red.
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Fig. 3. X-Gal stained mammary whole mounts (A,D-F) and their histological sections (B,C,G-I) of WAP-Cre/Rosa-lacZ double transgenic females at day 8 of the second gestation period (A-C) and in transplants of parous (D,E,G,H,I) and nulliparous (F) WAP-Cre/Rosa-lacZ epithelia into nulliparous wild-type recipients. Note that the parity-induced epithelial cells in involuted WAP-Cre/Rosa-lacZ animals serve as alveolar precursors in subsequent gestation cycles (A,B) but they remain essentially absent from larger ducts (C). X-Gal positive cells from involuted double transgenic mice contribute to ductal morphogenesis in transplants (D,E), and they are present in large ducts (G), small ducts (H) and terminal end buds (I). Control transplants from nulliparous double transgenic donors into wild-type recipients remain X-Gal negative (F) suggesting that the WAP-Cre transgene is not activated by the transplantation technique itself. LN, lymph node.
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Fig. 4. Rescue of the lactation-deficient phenotype after three successive post-partum periods (A,C,E) and their matching involution phases (B,D,F) in heterozygous prolactin receptor knockout mice that carry in addition the transgenic reporter constructs (WAP-Cre/Rosa-lacZ). Note the simultaneous increase in the number of parity-induced (X-Gal positive; blue) epithelial cells after each involution period and the reversion of the lactation-deficient phenotype and formation of normal secretory lobules after the third pregnancy (E). B, lower left, magnification of a selected area on the right to demonstrate the presence of a few X-Gal positive cells after the first reproductive cycle.
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Fig. 5. X-Gal staining of primary cultures of mammary epithelial cells (A-H,L), mammary fibroblasts (I), and a whole mount (J) and its corresponding histological section (K) of an outgrowth from transplanted dissociated cells of parous (involuted) WAP-Cre/Rosa-lacZ mice into the cleared fat pad of nulliparous wild-type recipients. Note the high proliferative capacity of blue cells derived from parous WAP-Cre/Rosa-lacZ mice and cultured for 48 (A) and 72 hours (B). Grown as a monolayer, these blue parity-induced cells do not differ in their morphology from other epithelial subtypes (C), and they can actively migrate away from epithelial colonies (D). Epithelial cells derived from nulliparous double transgenic mice and cultured for 48 (E,F) and 72 hours (G) are mostly X-Gal negative. The few blue cells detected in these early cultures of nulliparous controls do not expand even after 96 hours in culture (H). Note also that mammary fibroblasts of parous double transgenic animals are X-Gal negative (I). (J) Reconstitution of the ductal tree after transplantation of a small number of epithelial cells from parous, non-pregnant WAP-Cre/Rosa-lacZ mice into the cleared fat pad of nulliparous wild-type recipients (limiting dilution experiment; X-Gal whole-mount staining). (K) Histological section of J counterstained with nuclear Fast Red.
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© The Company of Biologists Ltd 2002
