Notch receptor, ligand, and target genes mRNA expression during postnatal mammary gland development (Raafat et al., revised version resubmitted). Conditional ablation of the Rbpj gene in progenitor cells of the mammary gland disrupts cell fate decisions and pregnancy-induced development of the gland. Little is known, however, about the timing and levels of expression of members of the Notch gene family, their ligands and affected target genes during normal mammary gland development. Therefore, we determined the timing and levels of mRNA expression of the different Notch receptors, their ligands and their canonical target genes during mouse postnatal mammary gland development. The levels of Notch1, Notch2 and Notch3 mRNA in the mammary gland increased as the mammary gland developed from 5 weeks of age to early pregnancy. Quantification of the expression of Notch receptor mRNAs in the mouse mammary gland during development revealed that Notch3 mRNA is the most abundant. Low levels of Notch4 mRNA were detected throughout various stages of mammary gland development. The decreasing levels of Notch receptor mRNAs in the mammary gland late in pregnancy is associated with decreased Rbpj dependent Notch signaling which allows for lobular alveolar development. The expression pattern and levels of mRNA for the Notch ligands is complex. Among these ligands, the levels of Jagged1 and Dll3 mRNA are the highest throughout mammary gland development. In one study of normal human mammary tissue, using in situ mRNA: mRNA hybridization, Jagged1 mRNA was detected in the normal ductal myoepithelium and a high level of Notch3 mRNA was detected in the adjacent ductal luminal epithelium. Immunohistochemical analysis of these reduction mammoplasties demonstrated that Notch1, Notch3, Jagged1 and Jagged2 proteins were in the lobular epithelium and not in the surrounding adipose tissue, whereas in our study of mice, Notch3 could be detected in the stroma as well as the luminal epithelium. This suggests that there are species differences in the expression patterns of Dll3 and Notch3 in the mammary gland. Canonical Notch signaling is mediated through the interaction of Notch ICD with the transcription repressor/activator Rbpj, the primary transcriptional mediator of Notch signaling. Interestingly, the expression level of Rbpj mRNA peaks in 5-day pregnant mammary glands in parallel with the induction of Notch signaling during pregnancy. Activation of Notch target genes should represent a reliable readout of Notch/Rbpj signaling activity. Known targets of Notch signaling include members of the Hes/Hey gene family that encode transcriptional repressors. The levels of Hey2 mRNA, at any given point during postnatal development of the mammary gland, are two orders of magnitude higher than Hey1, Hes1, or Hes5 mRNA levels. The functional significance of the complex pattern of temporal and spatial expression of components of the Notch signaling pathway in the different compartments of the mammary gland await the development of new mouse strains in which particular Notch genes or Notch ligand genes are conditionally ablated or in which the expression of a reporter gene is put under the transcriptional control of the particular Notch gene or Notch ligand gene promoter. Rbpj Conditional Ablation Reveals Distinct Functions for Notch4/Int3 in Mammary Gland Development and Tumorigenesis (Raafat et al. Oncogene In Press). The MMTV LTR -Int3 and Wap-Int3 transgenic mice exhibit two phenotypes with 100% penetrance: lack of mammary alveolar/lobular development during pregnancy and mammary tumor development. To determine whether these phenotypes result from the canonical Int3/Rbpj signaling pathway, we conditionally deleted exons 6 and 7 of the Rbpj gene in the presence of the mammary gland specific Wap-Cre transgene. Exons 6 and -7 encode DNA-binding and Notch-binding domains; and loss of these exons results in the complete loss of Rbpj mediated Notch signaling. Through a series of genetic crosses between Wap-Cre, Rbpj flox/flox and Wap-Int3 mice, female mice with the following genotypes were obtained: Wap-Cre+/Rbpj -/-/ Wap-Int3 (Wap-Int3/Rbpj knockout); Wap -Cre+/Rbpj -/+/Wap-Int3 (Wap-Int3/Rbpj heterozygous); and Rbpj flox/flox/ Wap -Int3 (Wap-Int3/Rbpj control). Ablation of Rbpj early in the postnatal development of the mammary gland prevents alveolar lobular development and leads to the accumulation of basal cells. Interestingly the Wap-Cre+/Rbpj -/- females that were used in the generation of the Rbpj knockout mice have no detectable phenotype with regard to mammary gland development. This suggests that the contribution of endogenous Notch/Rbpj signaling to mammary alveolar/lobular development is completed prior to the expression of the Wap-Cre transgene. Wap-Int3 expression blocks mammary alveolar/lobular development. However, the effects of Wap-Int3 expression on mammary gland development are not manifested in Wap-Int3/Rbpj knockout females. The morphology of the mammary gland from day1 lactating females is very similar to the normal FVB/N mammary gland and ?-casein can be detected in the alveolar/lobular structures. Morphological analysis of mammary glands, revealed the presence of focal hyper-proliferative lesions arising within the mammary ducts of Wap-Int3/Rbpj knockout, -heterozygous, and -control females. Irrespective of the genotype, the number of the lesions averaged 24/gland. By the third pregnancy, 100% of the Wap-Int3/Rbpj heterozygous and Wap-Int3/Rbpj control mice developed mammary tumors. However, tumor free survival was longer in the Wap-Int3/Rbpj knockout females, 80% of them developed tumors after 3 pregnancies and after 4 pregnancies 100% had mammary tumors. Thus although the loss of Rbpj function appears to have no effect on Int3 induced mammary tumorigenesis it may affect the rate of tumor cell proliferation. Taken together our data are most consistent with the conclusion that constitutive Int3 signaling blocks mammary alveolar development through a Rbpj dependent pathway and that Int3 induced mammary tumorigenesis or mammary tumor growth occurs as a consequence of Int3 signaling that is not dependent on the canonical Rbpj dependent pathway.
