Expression of Notch receptors, ligands and target genes during development of the mouse mammary gland. Notch genes play a critical role in mammary gland growth, development and tumorigenesis. In the present study we have quantitatively determined the levels and mRNA expression patterns of the Notch receptor genes, their ligands and target genes in the postnatal mouse mammary gland. The steady state levels of Notch3 mRNA are the highest among receptor genes, Jagged1 and Dll3 mRNA levels are the highest among ligand genes and Hey2 mRNA levels are highest among expressed Hes/Hey target genes analyzed during different stages of postnatal mammary gland development. We have used an immunohistochemical approach with antibodies specific for each Notch protein, to show that Notch gene expression in mammary epithelial cells is temporally regulated during normal mammary gland development in the FVB/N mouse. For instance in the peripubertal mammary gland, terminal end buds (TEBs) that drive ductal morphogenesis, are comprised of an outer cap cell layer enriched in mammary stem cells and inner body cells fated to the luminal compartment (Daniel et al., 1987). High levels of Notch4 and Hey2 expression were detected in TEB cap cells, which are the most proliferating population at this stage (Fendrick et al., 1998). The cap cell layer was devoid of Notch1, Notch2 and Notch3 suggesting that Notch4-positive cells constitute the major pool of proliferating cells at this developmental stage. We have also examined the effect of ovariectomy on Notch receptor and ligand gene expression in the mammary gland. The loss of ovarian hormones is associated with changes in the levels of Notch receptor mRNAs (Notch2 higher and Notch3 lower) and ligand mRNAs (Dll1 and Dll4 are higher, whereas Dll3 and Jagged1 are lower) in the mammary gland of ovariectomized mice compared to intact mice. Down regulation of Notch2, Dll1 and Dll4 in the intact mammary gland compared to mammary glands from ovariectomized mice suggests that certain regulatory sequences and/or chromatin structures are playing a role in the regulation of these genes (Muller et al., 2009, Strom et al., 2000). A primary consequence of ovariectomy is a loss of estrogen production, a known positive, or in some cases negative, regulator of gene transcription. Based on the effects of ovariectomy on transcription of some Notch receptor and ligand genes, we examined promoter regions for the presence of estrogen responsive elements (ERE). Although the EREs that were detected in Notch receptor and ligand genes were imperfect as compared to the perfect ERE palindrome (5'-GGTCANNNTGACC-3') (Lin et al., 2004), there is strong evidence that EREs can function in more loosely structured sequence motifs (Klein-Hitpass et al., 1986). Duplication of ERE half-palindromes in Notch1, Notch2, Notch3, Dll3, Dll4 and Jagged2 promoters provide a structural basis for estrogenic regulation of these genes. Transforming acidic coiled-coil protein-3 (Tacc3) acts as a negative regulator of Notch signaling through binding to CDC10 /Ankyrin. We have identified the transforming acidic coiled-coil protein-3 (Tacc3) as a binding partner for Notch4/Int3 and were able to show that it binds to the intracellular domain (ICD) of all members of the Notch receptor family. Members of the Tacc family reside at the centrosomes and associates with microtubules. Recent studies suggest that Tacc3 also contributes to the regulation of gene transcription. Tacc3 specifically interacts with the Notch4/Int3 CDC10/Ankyrin repeats and to a lesser extent, with residues C-terminal to these repeats in the ICD. The affinity of Tacc3 binding to Notch4/Int3 ICD is similar to that between Rbpj and Notch4/Int3 ICD. Notch4/Int3 ICD-Tacc3 interaction results in the inhibition of transcription from a Hes1-Luciferase reporter vector in COS-1 cells. The inhibition was reversed in these cells by increasing the levels of Rbpj. Dual label immunofluorescence of mouse mammary tissue shows Tacc3 co-localizes with the Notch3 ICD. We measured the expression levels of Tacc and Notch mRNA in the mammary gland. Interestingly, steady state levels of Tacc3 mRNA are highest in the mammary glands of 5 wk old nulliparous and pregnant females when the gland is in active growth phase and low in lactating and early involuted mammary glands. Tacc3 binds to the ICD of each member of the Notch receptor family. To demonstrate that Tacc3 co-localizes in vivo with a member of the Notch receptor family we have used double-labeled immuno-histochemical analysis of Tacc3 and Notch3 in mammary gland tissue of 5 wk old nulliparous, 5 day pregnant and 7 day involuted mice. Tacc3 and Notch3 showed nuclear localization in the 7 day involuted mammary gland. However, Notch3 and Tacc3 showed cytoplasmic staining in 5 wk nulliparous and 5-day pregnant mammary glands. Since all cells were positive for Tacc3 in the tested mammary developmental stages, it was possible that the Tacc3 expression per cell was different. To address this question, Tacc3 immunofluorescein intensity was quantified. This analysis showed significant differences in Tacc3 cellular content in the 5 wk nulliparous and 5 day pregnant mammary gland versus the 7 day involuted mammary gland. To determine whether the physical association between Tacc3 and Notch would occur in normal tissue, mammary and lung tissue were collected from 10-week old FVB mice. Tissue lysates were subjected to immunoprecipitation with Tacc3 or Notch4 antibodies. The immunoprecipitates were probed with anti-Tacc3 or anti-Notch4 antibodies. Immunostaining analysis revealed a predominant interaction between Tacc3 and Notch4. To further investigate the effect of Taac3 and Notch interaction on Notch signaling, NIH3T3 cells were transfected with increasing levels of Tacc3 -siRNA (3, 6, and 12 mug, respectively). Quantitative RT-PCR analysis of Tacc3 expression showed decrease in the Tacc3 expression as the Tacc3-siRNA increased. At 12 mug of Tacc3-siRNA caused 85% average decrease in Tacc3. Interestingly, Hey2 expression increased in the same cells as the Tacc3 expression decreased in a dose-dependent pattern. At 12mug of Tacc3-siRNA, Hey2 mRNA expression increased 4 fold. Taken together, these results suggest that Tacc3 is a negative regulator of the Notch signaling pathway.
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