s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue - PubMed (original) (raw)

s-SHIP promoter expression marks activated stem cells in developing mouse mammary tissue

Lixia Bai et al. Genes Dev. 2010.

Abstract

Mammary stem cells (MaSCs) play critical roles in normal development and perhaps tumorigenesis of the mammary gland. Using combined cell markers, adult MaSCs have been enriched in a basal cell population, but the exact identity of MaSCs remains unknown. We used the s-SHIP promoter to tag presumptive stem cells with GFP in the embryos of a transgenic mouse model. Here we show, in postnatal mammary gland development, that GFP(+) cap cells in puberty and basal alveolar bud cells in pregnancy each exhibit self-renewal and regenerative capabilities for all mammary epithelial cells of a new functional mammary gland upon transplantation. Single GFP(+) cells can regenerate the mammary epithelial network. GFP(+) mammary epithelial cells are p63(+), CD24(mod), CD49f(high), and CD29(high); are actively proliferating; and express s-SHIP mRNA. Overall, our results identify the activated MaSC population in vivo at the forefront of rapidly developing terminal end buds (puberty) and alveolar buds (pregnancy) in the mammary gland. In addition, GFP(+) basal cells are expanded in MMTV-Wnt1 breast tumors but not in ErbB2 tumors. These results enable MaSC in situ identification and isolation via a consistent single parameter using a new mouse model with applications for further analyses of normal and potential cancer stem cells.

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Figures

Figure 1.

Figure 1.

GFP expression occurs in cap cells of the TEBs at puberty. (A) TEBs (arrows) in carmine whole-mount-stained 5-wk mammary tissue. (B) GFP+ TEBs (green, arrows) and blood vessels (green, arrowheads) in 4-wk transgenic whole-mount mammary tissue. (C) One section of a 4-wk TEB. Cap cells are GFP+ (short arrows), and some GFP+ cap cells are among the GFP− body cells (long arrows). (Red) Phalloidin-Alexa 594; (blue) DAPI. (D) GFP+ cap cells (green) are also p63+ (red); shown in enlargement (left panel; GFP and p63 only), and separately (middle two panels). Arrows mark the cap cell layer. (E) Cap cells (green) are SMA+ (red). (F) Most GFP+ cap cells are Ki67+ (red). (G) Some GFP+ cap cells were labeled with BrdU (red) within 4 h of BrdU injection. Bars: A,B, 1 mm; C,D,F,G, 100 μm; C (enlarged), E, 50 μm. (H) Histogram shows that 93.8% ± 2.4% of GFP+ cells in TEBs are Ki67+, and 34.6% ± 5.9% of GFP+ cells were labeled with BrdU during a 4-h exposure. Data represent mean ± SD; n = 20 TEBs.

Figure 2.

Figure 2.

Characterization of GFP+ cap cells in puberty. (A) Histogram shows the percentage of lin−GFP+CD49fhigh cap cells in total lin− puberty mammary cells. Results represent mean ± SD of four mice for each group. (B–E) Flow cytometric analysis shows that GFP+CD49fhigh cap cells are CD29high (B), Sca-1−/low (C), CD133− (D), and CD61+ (E). (F) Side population analysis shows that puberty GFP+ cells are depleted in the side population. (G) RT–PCR shows s-SHIP mRNA in sorted GFP+ cells but not in GFP− cells. (H,I) Single GFP+CD49fhigh cap cells form alveolar-like colonies in Matrigel; photographed with a Nikon microscope in situ in Martrigel (phase and GFP) (H), or removed from Matrigel and examined for GFP by confocal microscopy (I). (J) K14+ cells (red) comprise the outer layer of the colony (DAPI-stained for nuclei; blue). Bars: H,J, 20 μm; I, 50 mm. (K) Histogram shows the colony-forming capacity of lin−GFP+CD49fhigh and lin−GFP− cells in Matrigel. Data represent mean ± SD of five independent experiments.

Figure 3.

Figure 3.

Characterization of transplantation outgrowths from puberty GFP+ cap cells. (A) GFP+ TEBs (green, arrow) in an outgrowth from 40 GFP+ cap cells; enlarged TEBs are shown in the inset. (*) Approximate injection site. (B) Carmine alum-stained ductal outgrowths from four (three to eight) cells, or one single GFP+ cap cell (inset), respectively. (C) X-gal staining shows the TEBs (arrowheads) in a second serial outgrowth originated from eight (six to 10) primary GFP+LacZ+ cap cells. (D,E) Outgrowth TEBs consist of GFP+ SMA+ cap cells (D) and E-cad+ body cells (E). (F–I) Pregnancy-induced lobulo–alveolar formation in lactating outgrowths (lactation day 0.5) derived from 40 GFP+LacZ+ cap cells. (F) X-gal whole-mount staining shows lobulo–alveolar structures of outgrowth. (G) Frozen section of outgrowth lobulo–alveoli stained with X-gal (blue) and nuclear fast red (red). (H) Antibody staining for milk protein (arrows) in alveolar lumen. (Red) Milk; (green) phalloidin; (blue) DAPI. (I) Outgrowth lobulo–alveoli are composed of SMA+ myoepithelial cells (arrowheads) and E-cad+ alveolar luminal cells. Bars: A,B,F, 1 mm; B (inset), C, 0.25 mm; D,E,G–I, 50 μm.

Figure 4.

Figure 4.

Identification and characterization of GFP+ cells in alveolar buds at pregnancy. (A–D) Alveolar buds in P11.5 transgenic mammary tissues. Carmine whole-mount staining shows alveolar buds along the ductal network (A), GFP expression (green) in the apical alveolar buds, and some blood vessels (B). Frozen sections show that GFP is expressed mainly in the peripheral cells, especially the tips of the alveolar buds (C,D), and some GFP+ cells are seen in the inner layer of the alveolar buds (D). (E) GFP+ cells in P2.5 mammary tissue are SMA+ (red). (F) Most GFP+ cells in P7.5 alveolar bud are Ki67+ (red). (G) Histogram shows that 91.5% ± 5.9% of GFP+ cells at pregnancy are Ki67+, and 27.7% ± 10.9% of GFP+ cells were labeled with BrdU within 4 h of injection. Data represent mean ± SD; n = 20 alveolar buds. (H) Flow cytometric analysis of lin− mammary cells from P5.5 mammary tissues. GFP+ cells consist of CD49fhigh alveolar bud cells (gate) and CD49f −/low blood vessel cells. (I) Histogram shows the percentage of GFP+CD49fhigh alveolar bud cells in the total lin− population during pregnancy. Results represent mean ± SD of four mice for each group. (J) Carmine alum staining of an outgrowth from 100 pregnancy GFP+ cells shows the TEBs (arrowheads) at the ductal ends. (K) GFP+ TEBs in the outgrowth of 50 pregnancy GFP+ cells. (*) Approximate injection site. Bars: A,B, 200 μm; C,D,F, 20 μm; E, 50 μm; J,K, 1 mm.

Figure 5.

Figure 5.

Comparison of GFP+ mammary epithelial cells with previously identified lin−CD24modCD49fhigh and lin−CD24modCD29high MaSC phenotypes. (A) Mammary cells from puberty (4w + 4d), adult virgin (6m), and pregnancy (P8.5d, 6m) were stained and analyzed simultaneously. The left panels show that the distributions of lin− mammary cells from different developmental stages based on CD24 and CD49f expression level are quite different. Unlike in virgin mammary cells, there are no clearly distinguishable CD24highCD49flow and CD24modCD49fhigh populations in puberty (4w + 4d) lin− mammary cells. The right panels show that GFP+ epithelial cells account for 9.0% and 10.8% of the overlapped lin−CD24modCD49fhigh cells in puberty (4w + 4d) and in pregnancy (P8.5d), respectively. Lin−CD24modCD49fhighGFP+ cells are not present in virgin transgenic mammary cells. (B) Mammary cells from puberty (4w + 1d), adult virgin (6m), and pregnancy (P8.5d, 6m) were stained and analyzed simultaneously. The left panels show the differences among the distribution of lin− mammary cells from different stages according to CD24 and CD29 expression. Clearly distinguishable CD24highCD29low and CD24modCD29high populations do not exist in puberty (4w + 4d) lin− mammary cells. The right panels show that GFP+ epithelial cells represent 22.4% and 4.0% of the overlapped lin−CD24modCD29high cells in puberty (4w + 1d) and pregnancy (P5.5d), respectively. (C) lin−CD24+CD49fhighGFP+ and lin−CD24+CD49fhighGFP− cells were sorted from pubertal mammary glands of 5-w + 1-d-old transgenic mice (shown in the dot plot), and were cultured in Matrigel or transplanted into cleared fat pads. The histogram shows the colony-forming capability of lin−CD24+CD49fhighGFP+ cells is significantly higher than that of lin−CD24+CD49fhighGFP− cells. Data represent mean ± SD of five independent experiments. The table shows lin−CD24+CD49fhighGFP+ cells possess significantly higher regenerative potential than lin−CD24+CD49fhighGFP− cells upon transplantation. The latter GFP− population may contain dormant MaSCs.

Figure 6.

Figure 6.

GFP expression in breast tumors. (A,B) GFP is expressed in Wnt1 tumors (A) and GFP+ cells are SMA+ (B). (C,D) No GFP expression was detected in ErbB2 tumors (C), and no SMA+ cells were detected in ErbB2 tumors (D). Bars, 50 μm.

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References

    1. Alexander CM, Puchalski J, Klos KS, Badders N, Ailles L, Kim CF, Dirks P, Smalley MJ 2009. Separating stem cells by flow cytometry: Reducing variability for solid tissues. Cell Stem Cell 5: 579–583 - PMC - PubMed
    1. Asselin-Labat ML, Shackleton M, Stingl J, Vaillant F, Forrest NC, Eaves CJ, Visvader JE, Lindeman GJ 2006. Steroid hormone receptor status of mouse mammary stem cells. J Natl Cancer Inst 98: 1011–1014 - PubMed
    1. Asselin-Labat ML, Sutherland KD, Barker H, Thomas R, Shackleton M, Forrest NC, Hartley L, Robb L, Grosveld FG, van der Wees J, et al. 2007. Gata-3 is an essential regulator of mammary-gland morphogenesis and luminal-cell differentiation. Nat Cell Biol 9: 201–209 - PubMed
    1. Barker N, van Es JH, Kuipers J, Kujala P, van den Born M, Cozijnsen M, Haegebarth A, Korving J, Begthel H, Peters PJ, et al. 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449: 1003–1007 - PubMed
    1. Bissell MJ, Labarge MA 2005. Context, tissue plasticity, and cancer: Are tumor stem cells also regulated by the microenvironment? Cancer Cell 7: 17–23 - PMC - PubMed

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