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, 5 (8), e11947

Redefining the Expression and Function of the Inhibitor of Differentiation 1 in Mammary Gland Development

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Redefining the Expression and Function of the Inhibitor of Differentiation 1 in Mammary Gland Development

Radhika Nair et al. PLoS One.

Abstract

The accumulation of poorly differentiated cells is a hallmark of breast neoplasia and progression. Thus an understanding of the factors controlling mammary differentiation is critical to a proper understanding of breast tumourigenesis. The Inhibitor of Differentiation 1 (Id1) protein has well documented roles in the control of mammary epithelial differentiation and proliferation in vitro and breast cancer progression in vivo. However, it has not been determined whether Id1 expression is sufficient for the inhibition of mammary epithelial differentiation or the promotion of neoplastic transformation in vivo. We now show that Id1 is not commonly expressed by the luminal mammary epithelia, as previously reported. Generation and analysis of a transgenic mouse model of Id1 overexpression in the mammary gland reveals that Id1 is insufficient for neoplastic progression in virgin animals or to prevent terminal differentiation of the luminal epithelia during pregnancy and lactation. Together, these data demonstrate that there is no luminal cell-autonomous role for Id1 in mammary epithelial cell fate determination, ductal morphogenesis and terminal differentiation.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Id1 expression in the mouse mammary gland.
Santa Cruz SC-488 polyclonal antibody does not specifically recognise Id1. Mammary glands from 10 week old virgin wildtype (A) and Id1-null (B) mice were immuno-stained for Id1 using SC-488. Note the non-specific positive staining of epithelial cells regardless of genotype (inset). A spontaneous p53-null mammary tumor was immunostained for Id1 using SC-488 (C) or BCH-1/#37-2 monoclonal antibody (D). (E) BCH-1/#37-2 monoclonal antibody was used to immunostain for Id1 in Id1-transgenic mammary glands, or spleen taken from a wildtype (WT) or Id1-null mouse. Id1-positive cells in the spleen are endothelium. (F) Id1 immunostaining (BCH-1/#37-2) was conducted on mouse mammary glands at various stages of mammary gland development: 5 weeks old, 12 week old virgin, 4 days post-coitus (dpc), 18 dpc, lactating 4 days postpartum (dpp) and lactating 15 dpp. Arrows indicate examples of Id1-positive cells.
Figure 2
Figure 2. Generation of a mammary-specific conditional Id1-transgenic mouse.
Mice from TRE-Id1 lines #3 or #10 were bred to MTB mice and administered dox chow for 5 days. (A) Mammary gland extracts were western blotted for Id1. The lower band is endogenous Id1 while the upper band corresponds to the Id1-HA transgene product. (B) Mammary glands or spleen were harvested and stained with anti-HA monoclonal antibody to demonstrate transgene expression in the luminal, but not in the myoepithelium (arrow) or non-epithelial organs such as spleen. TRE-Id1 line #10 gave identical results.
Figure 3
Figure 3. Ectopic Id1 expression is not sufficient to perturb virgin mammary gland development.
Mice from TRE-Id1 lines #3 and #10 or TRE-Myc were bred to MTB mice and administered dox chow from 3 to 10 weeks of age to induce transgene expression. Mammary morphogenesis was analysed by carmine alum whole mounts (above) and by hematoxylin and eosin staining of sections from formalin-fixed, paraffin embedded glands (below). The histology of Id1 transgenic glands was unaffected by Id1 transgene expression during virgin mammary development, while control myc-transgenic glands display a hyperplastic phenotype. Images are representative of at least 5 mice per group, across two independent transgenic lines.
Figure 4
Figure 4. Id1 does not control terminal differentiation in the mammary gland.
TRE-Id1/MTB double transgenic (DT) females or single transgenic (ST) controls were placed on dox chow from 6 weeks. (A–D) At ∼8–20 weeks, females were mated to wildtype males. At 16 days post coitus, mammary glands were harvested and analysed by carmine whole mount (A), sectioned for histology (B) or RNA extracted and analysed for Id1 (C) and β-casein gene expression (D). To determine whether TRE-Id1/MTB bitransgenic mice can lactate, pups were weighed after birth for up to 8 days and the average weight per litter compared to or single transgenic control mice (E).

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