Hematoxylin/eosin stained sections of mammary glands.

<p>Mammary glands from D8P, D15P, and D1L <i>Ndst1</i>^f/f<i>MMTVCre</i>⁻ (<b>A–C, respectively</b>) and <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ (<b>D–F, respectively</b>) mice were stained with hematoxylin/eosin. No difference in glandular morphology was noted at D8P, but differences in ductal density occurred at D15P, which is quantified in (<b>G</b>). The difference in density between mutant and wildtype increased dramatically by D1L. Bars  = 125 µm.</p

Altered expression of heparan sulfate in <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ mammary epithelia.

<p>(<b>A</b>) Frozen sections of mammary glands were incubated with biotinylated FGF2, which binds to heparan sulfate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Bai1" target="_blank">[55]</a>. Binding of FGF2 was detected with streptavidin-HRP (brown stain). In wildtype <i>Ndst1</i>^f/f<i>MMTVCre</i>^– glands FGF2 binds to the basement membrane surrounding the epithelial ducts (arrowheads). The right panel magnifies the boxed region, revealing the sharp staining of the basement membrane underlying the epithelial cells. FGF2 also binds to the matrix surrounding the fat pad adipocytes (white arrowheads). Mutant <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ glands retain FGF2 binding around fat pad adipocytes, but binding to the basement membrane was greatly reduced. The lower right panel magnifies the boxed region. The bar in the left panels  = 50 µm, right panels 12.5 µm. (<b>B</b>) Heparan sulfate was isolated from [6-³H]glucosamine labeled mammary epithelial cells derived from <i>Ndst1</i>^f/f<i>MMTVCre</i>^– and <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ mice and degraded with nitrous acid <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Shively1" target="_blank">[58]</a>. The individual oligosaccharides were separated by gel filtration chromatography and the area under the peaks was used to determine the extent of N-sulfation of the chains <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010691#pone.0010691-Bame1" target="_blank">[59]</a>. dp, degree of polymerization. Inset: Graph of comparison of areas under the curves.</p

Characterization of the lactational defect in <i>Ndst1</i>-deficient mammary glands.

<p>Quantitative RT-PCR and Western blotting were used to characterize lactational capacity of <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ females. (<b>A</b>) RNA isolated from d1L <i>Ndst1</i>^f/f<i>MMTVCre</i>^– and <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ mammary glands was analyzed by quantitative RT-PCR for the expression of whey acidic protein (WAP), β-casein, and keratin 18, genes specifically expressed by mammary epithelial cells. Data was normalized to the expression of GAPDH transcripts present in the sample. (<b>B</b>) Western blotting with antibodies to keratin 7 in d1L glands further confirmed that the <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ mammary glands have a reduced population of epithelia. (<b>C</b>) Western blotting with antibodies to mouse milk from d1L glands shows that milk production was diminished in <i>Ndst1</i>^f/f<i>MMTVCre</i>⁺ glands. Blotting of d14P extracts showed that the antibodies were selective for milk protein except for a minor band at 67 kDa.</p