Protein Markers for Mammary Differentiation (MMD)

Recently, we have described the use of several protein markers that exhibit distinct patterns of expression in the mammary gland.

Using a variety of knockout mouse models that exhibit mammary gland phenotypes, we have further demonstrated the use of these markers for the proteotyping of mammary gland development. So far, we have used these markers in the proteotyping of mammary gland phenotypes observed in Jak2, PrlR and Stat5 KO mammary transplants; IKKalpha KO mice; conditional Stat3 KO mice; conditional ErbB4 KO mice; inhibin betaB KO mice; DGAT1-null mice; and DDR1-null mice.

These markers can be applied to the proteotyping of mammary gland development in mouse model system and as such represent a powerful tool for the analysis of mammary gland development.

Profiling of protein markers during mammary gland development


                                                        6 week virgin             preg day 12             lactation day 1


AQP5:   The water transporter, aquaporin 5 (AQP5), is present on the apical membrane of ductal cells during virgin development (A). Interestingly, AQP5 was not detected during pregnancy even in ductal structures (B). No apical AQP5 was apparent at lactation (C)

NKCC1:   The Na-K-Cl cotransporter, NKCC1, is present at high levels on the basolateral membrane of ductal mammary epithelial cells in virgin animals (D) and is downregulated in developing alveoli and ductal structures at pregnancy (E). During lactation, high levels of NKCC1 protein are only observed in a few cells within alveoli (F).

Npt2b:   The Na-Pi cotransporter isoform, Npt2b, is not detectable in mammary ductal cells in virgin animals (G) or during early- to mid-pregnancy (H). Npt2b is first observed at late pregnancy (data not shown) and throughout lactation (I). During involution, Npt2b is lost within 48h (data not shown).

Profiling of protein markers in knockout mouse models

In the absence of the PrlR, Jak2 or Stat5, mammary epithelial cells fail to proliferate, show histological evidence of ductal-like structures and a failure to fill the mammary fat pad at parturition. The images below demonstrate that high levels of the basolateral ductal marker, NKCC1, are maintained and AQP5 is also present on the apical membrane of PrlR-, Jak2- and Stat5-null epithelium at lactation. Furthermore, the induction of a secretory marker, Npt2b, is not apparent in these mouse models. Taken together, and in support of other data, these results confirm that signaling via the PrlR-Jak2-Stat5 pathway is necessary for functional differentiation of mammary epithelial cells during pregnancy.

                                    wild-type                       PrlR-null                       Jak2-null                      Stat5-null





Conditional deletion of Stat3 from mammary epithelium results in a failure of the gland to involute upon weaning. In wild type animals Npt2b was not detectable on the apical membrane by day 2 of involution but was present in the lumen, probably reflecting shedding of the apical membrane. By involution day 6, wild type mammary epithelium is almost compeltely remodelled and no Npt2b staining is apparent.

In contrast, when Stat3 is selectively deleted from the epithelial compartment, Npt2b remains detectable on the apical membrane at day 2 of involution even though there is some apical membrane shedding. By day 6 of involution, the gland fails to undergo remodelling and the alveolar lumen contain a significant amount of immunoreactive Npt2b similar to that seen at day 2 of involution in wild type animals.

Therefore, using the Npt2b marker, we were able to demonstrate that delayed involution was associated with a significant delay in the disappearence of apical Npt2b.

                                          lactation day 10                   involution day 2                   involution day 6



In an attempt to establish the overall lactational competence of the Stat3-null glands, an experiment was devised in which animals were allowed to lactate for 10 days, involute for 6 and then suckled for a further 5 days. In wild type animals, the gland had remodelled by involution day 6 and pups were unable to thrive when placed with the lactating dam. Interestingly, the majority of pups placed with Stat3 fl/fl;WC involuted dams survived and there was evidence of milk in their stomachs. This observation corresponded with the re-induction of apical Npt2b, suggesting that Npt2b is an ideal protein marker for exmaining the lactational competence of the animal.


                                                              involution day 6                        resuckled day 5

Relevant references

1. Shillingford, J.M., Miyoshi, K., Robinson, G.W., Bierie, B., Cao, Y., Karin, M. and Hennighausen, L.(2003). Proteotyping of mammary tissue from transgenic and gene knockout mice with immunohistochemical markers. A tool to define developmental lesions J. Histochem. Cytochem. 51:555-565.

2. Humphreys, R.C., Bierie, B., Zhao, L., Raz, R., Levy, D. and Hennighausen L. (2002). Deletion of Stat3 blocks mammary gland involution and extends functional competence of the secretory epithelium in the absence of lactogenic stimuli. Endocrinology 143:3641-3650.

3. Shillingford, J.M., Miyoshi, K., Flagella, M., Shull, G.E. and Hennighausen, L. (2002). Mouse mammary epithelial cells express the Na-K-Cl cotransporter, NKCC1: characterization, localization, and involvement in ductal development and morphogenesis. Mol. Endocrinol. 16:1309-1321.

4. Shillingford, J.M., Miyoshi, K., Robinson, G.W., Grimm, S.L., Rosen, J.M., Neubauer, H., Pfeffer, K. and Hennighausen, L. (2002). Jak2 is an essential tyrosine kinase involved in pregnancy-mediated development of mammary secretory epithelium. Mol. Endocrinol. 16:563-570.

5. Miyoshi, K., Shillingford, J.M., Smith, G.H., Grimm, S.L., Wagner, K.U., Oka, T., Rosen, J.M., Robinson, G.W. and Hennighausen, L. (2001). Signal transducer and activator of transcription (Stat) 5 controls the proliferation and differentiation of mammary alveolar epithelium. J. Cell. Biol. 155:531-542.

  For additional information contact Jonathan Shillingford

last update: August 5th 2003


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