The role of Sox9 in mouse mammary gland development and maintenance of mammary stem and luminal progenitor cells - PubMed (original) (raw)

The role of Sox9 in mouse mammary gland development and maintenance of mammary stem and luminal progenitor cells

Gautam K Malhotra et al. BMC Dev Biol. 2014.

Abstract

Background: Identification and characterization of molecular controls that regulate mammary stem and progenitor cell homeostasis are critical to our understanding of normal mammary gland development and its pathology.

Results: We demonstrate that conditional knockout of Sox9 in the mouse mammary gland results in impaired postnatal development. In short-term lineage tracing in the postnatal mouse mammary gland using Sox9-CreER driven reporters, Sox9 marked primarily the luminal progenitors and bipotent stem/progenitor cells within the basal mammary epithelial compartment. In contrast, long-term lineage tracing studies demonstrate that Sox9+ precursors gave rise to both luminal and myoepithelial cell lineages. Finally, fate mapping of Sox9 deleted cells demonstrates that Sox9 is essential for luminal, but not myoepithelial, lineage commitment and proliferation.

Conclusions: These studies identify Sox9 as a key regulator of mammary gland development and stem/progenitor maintenance.

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Figures

Figure 1

Figure 1

Deletion of Sox9 results in mammary gland defects in 3 and 5 weeks old mice. Whole mount analysis of mammary glands from control (MMTV-Cre; Sox9wt/wt) and Sox9 cKO (MMTV-Cre; Sox9fl/fl) mice. Mammary glands were harvested at 3, 5, 8 weeks, mid-pregnancy, lactation day 1, and lactation day 10 and stained with carmine alum and analyzed under the microscope.

Figure 2

Figure 2

Sox9 deleted cells are lost over time. Immunostaining of control (MMTV-Cre; Sox9wt/wt) and Sox9 cKO (MMTV-Cre; Sox9fl/fl) mouse mammary glands cut sections (3, 5, 8 weeks old mice) with Cre antibody (red). Green and blue represent anti-SMA and DAPI staining, respectively.

Figure 3

Figure 3

Short-term linage tracing shows that Sox9 is exclusively expressed in luminal cells. (A). Sox9-CreERT2; CAG-EGFP mice were injected with tamoxifen. 72 hr after injection, the mice were sacrificed for mammary gland staining and FACS analysis. Sections of the mouse mammary gland were stained with GFP (green) and myoepithelial cell marker (α-SMA) (red) antibodies and DAPI (staining nuclei, blue). (B) Flow cytometry analysis of CD24 expression of GFP-positive and -negative cells isolated from above tamoxifen treated mouse mammary glands. (C). Flow cytometry analysis of all CD24+ luminal cells (both GFP+ and GFP-) isolated from the above tamoxifen treated mouse mammary glands using CD61 and CD49f expression. This demonstrates that luminal cells of the mammary gland are comprised of CD61+ luminal progenitors and CD61- differentiated luminal cells. (D). CD61, CD49f flow cytometry analysis of only GFP+ cells isolated from above tamoxifen treated mouse mammary glands shows that GFP+ cells are mostly CD61+ luminal progenitors. (E) CD24, CD29 flow cytometry analysis of GFP positive cells isolated from above tamoxifen treated mouse mammary glands.

Figure 4

Figure 4

Long-term linage tracing shows that Sox9 expressing cells are capable of giving rise to both luminal and myoepithelial lineages (A). X-gal stained tissue section of adult virgin Sox9-CreERT2; R26R-LacZ mammary gland injected with TM and harvested after 4 weeks. Arrows point to reporter activity in myoepithelial cells. (B). GFP stained tissue section of adult virgin Sox9-CreERT2; CAG-EGFP mammary gland injected with TM and harvested after 4 weeks. Arrows highlight the progeny of Sox9 expressing cells within the myoepithelial layer (C). Flow cytometry analysis of CD24, GFP expression of GFP positive and negative cells isolated from above adult virgin mice mammary glands. (D). Flow cytometry analysis of CD61, CD29 expression of GFP+ cells isolated from above adult virgin mice mammary glands.

Figure 5

Figure 5

Non-inducible long-term linage tracing shows that Sox9 is expressed in luminal and myoepithelial cells. A non-inducible Sox9-Cre knock-in mouse model was used to assess the contribution of Sox9+ cells to mature lineages. Sox9-Cre mice were crossed with CAG-EGFP mice. Sox9-Cre; CAG-EGFP mammary gland cut sections were stained with anti-GFP (green) and α-SMA (red) antibodies. DAPI staining is blue. (A) GFP staining shows Sox9 expressing cells contribute to both luminal and myoepithelial lineages. (B) α-SMA (red) is used to highlight the myoepithelial cells.

Figure 6

Figure 6

Sox9 deleted luminal cells are lost over time. (A). LacZ staining of whole mount mammary glands from 5 week old Sox9 cKO (R26R; MMTV-Cre; Sox9fl/fl) mouse. (B). GFP in whole mount 8 week old Sox9 cKO (CAG-EGFP; MMTV-Cre; Sox9fl/fl) was visualized with fluorescence microscope. (C). Section of mammary glands from 5-week old Sox9 cKO (R26R; MMTV-Cre; Sox9fl/fl) mouse, stained with X-gal. (D). Section of mammary glands from 8 week old Sox9 cKO (CAG-EGFP; MMTV-Cre; Sox9fl/fl) mouse, immuno-labeling with anti-GFP (green) and anti-α-SMA (red) antibodies. (E). CD24, GFP FACS analysis of cells isolated from control (CAG-EGFP;MMTV-Cre; Sox9wt/wt) mouse mammary glands. (F). CD24, GFP Flow cytometry analysis of cells isolated from Sox9 deletion (MMTV-Cre; Sox9fl/fl; CAG-EGFP) mouse mammary glands.

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