Transgenic expression of truncated int3 in mammary tissue (WAP-int3) leads to impaired alveolar development
whole mount image of a mammary gland from a WAP-Int3 transgenic mouse at parturition that expresses a truncated int3 gene under the control of the mouse WAP gene promoter. Note the lack of alveolar development.
Summary
Transgenic mice expressing the cytoplasmic portion of the Int3 gene under control of the WAP gene promoter develop mammary tumors at complete penetrance in both breeding and virgin females. The tumor latency is significantly shorter (about one-half) in parous females than in nulliparous. This is thought to be due to the dependency of transgene expression from the WAP promoter which is very active in the secretory mammary epithelium during late pregnancy and lactation. Male WAP/Int3 mice exhibit no aberrant phenotypic features and are fertile. Mammary ductal growth and extension is normal in virgin female WAP/Int3 mice, but lobular development is severely curtailed during pregnancy. FVB/WAP/Int3 females are unable to lactate.
Note:
the truncated int3 has also been expressed under control of the MMTV-LTR (information).
Review on the role of int3 in development and neoplasia
Citations
Gallahan, D., Jhappan, C., Robinson, G., Hennighausen, L., Sharp, R., Kordon, E.C., Callahan, R., Merlino, G.,and Smith, G.H. (1996) Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis. Cancer Res. 56, 1775-1785, 1996.
Gertraud W. Robinson, Gilbert H. Smith, Daniel Gallahan, Andreas Zimmer, Priscilla A. Furth, Lothar Hennighausen. Understanding mammary gland development through the imbalanced expression of growth regulators. Developmental Dynamics, 206, 159-168.
Smith,G.H., Gallahan,D., Diella,F., Jhappan,C., Merlino,G. and Callahan,R. Constitutive expression of a truncated INT3 gene in mouse mammary epithelium impairs differentiation and functional development. Cell Growth & Different. 6: 563-577, 1995.
Background
Insertional mutation of the Int3 gene, a member of the Notch gene family, is frequently associated with primary mouse mammary tumors induced by the mouse mammary tumor virus (MMTV). A major consequence of these mutations is the production of a shortened 2.4 kb tumor-specific Int3 RNA transcript which encodes the entire intracellular domain of the Int3 protein. Previous studies have demonstrated that mammary gland development and function was severely impaired in transgenic mice expressing the truncated Int3 gene product from the MMTV viral promoter. Both mammary ductal growth and secretory lobule development was curtailed in these mice. These results were attributed to a gain of function modification of the Int3 gene which led to a restriction of cell fate selection in the affected mammary epithelial cells. To confirm and extend these findings, truncated Int3 was expressed from the whey acidic protein (WAP) promoter whose activity, unlike the MMTV-LTR, is restricted to the secretory mammary epithelial population. In transgenic mice carrying the WAP-Int3 construct, mammary ductal growth was unaffected in virgin females, but growth and differentiation of secretory lobules during gestation was profoundly inhibited. Coincidental with the block in lobular secretory differentiation, mammary dysplasia and tumorigenesis occurred in all breeding females by 25 weeks of age. In non-breeding WAP-Int3 females mammary tumor incidence also reached 100% but only after 70 weeks. The WAP-Int3 mammary tumors were highly malignant and most tumor-bearing females, irrespective of breeding history developed metastatic lung lesions. These results suggest that WAP-promoter targeted Int3 function is associated with mammary secretory cell differentiation and maintenance in this transgenic model. Consistent with the conclusion that WAP-driven truncated Int3 expression influenced only lobular differentiation and not ductal growth and extension during mammary gland development, transplants of WAP/Int3 gland into non-transgenic mammary fat pads produced complete mammary ductal outgrowths in virgin FVB/N mice but failed to develop secretory lobules when the females were impregnated.
Mammary phenotype
Severe retardation of lobular development in full term pregnant and post-partum females with a concommitant absence of ability to lactate (slide 4).
Mammary ducts in the transgenic virgin WAP-Int3 female completely filled the fat pad and appears normal (slide 2). Lobular development during mid and late pregnancy is severely retarded (slide 3).
ducts
normal development in mature females.
alveoli
Alveolar cell and ductal luminal cells poorly differentiated no secretory cytological development, absence of secretory droplets and milk fat globules at term. Multiple dysplastic regions along the lateral surface of ducts extending into the ductal lumina (slide 5).
gene expression
little, or no detectable RNA for casein, WAP; high expression of Int3 transgene in pregnant gland (slide 6)
Tumorigenesis
Mammary tumors appear in 100% of WAP-Int3 females, both breeders and virgins. Lung metastases are present in a high percentage of tumor-bearing WAP/Int3 females (8/8 breeding females and 5/11 virgin had lung metastasis). INSERT (Fig. 9 from Cancer Res. paper).
Transplantation studies
Implants of WAP-Int3 glands into mammary fat pads of virgin non-transgenic hosts divested of indigenous epithelium indicate the capacity of WAP-Int3 epithelium to grow normal ductal trees but they fail to produce functional lobules in non-transgenic pregnant hosts.
int3 expression under WAP control vs. the MMTV-LTR
There are several striking differences between the WAP/Int3 and the MMTV-LTR/Int3 transgenic model systems with respect to mammary gland growth and development. A full and completely branched mammary ductal system develops in virgin WAP/Int3 females and the WAP/Int3 ductal epithelium forms the appropriate intercellular connections necessary for a proper ductal morphology. Neither of these events is accomplished in the virgin MMTV-LTR/Int3 transgenic mammary glands (
Coincident with the limited lobular development of the WAP-Int3 mammary gland during pregnancy, dysplastic lesions appear throughout the gland. These lesions do not regress after weaning, but progress to frank carcinoma. Although mammary tumors also appear in 100% of non-breeding virgin WAP/Int3 females, the latent period for reaching 50% tumor incidence is more than double that required for the same incidence in breeders. In contrast, there is no significant difference in age of tumor onset between virgin and parous MMTV-LTR/Int3 females (manuscript in preparation, Smith et al ). This is probably due to the difference in the temporal pattern of transgene expression between the two lines.
There are two distinct patterns of growth and development in the mouse mammary gland. The first involves the penetration of the mammary fat pad by branching ductal morphogenesis and is under the control of ovarian hormones and local growth factors. The second involves the growth and expansion of the secretory lobules at the onset of pregnancy. This is dependent upon the hormonal status induced by pregnancy and continues until parturition. Each of these periods of expansion involves the production of epithelial progenitors committed to a specific developmental lineage (i.e., ducts and lobules) which are responsive to specific inductive and growth signals. In the MMTV-LTR/Int3 transgenic line, the transgene is expressed in both types of progenitors and their progeny and limits the capacity of each to fulfill its differentiative program. In the WAP/Int3 line, expression of the transgene is restricted to the pregnancy-induced secretory progenitor responsible for the fabrication of the alveolar structures and leads to an attenuation of their functional differentiation. Consistent with this posit, full term pregnant WAP/Int3 glands possess lobular epithelium that is strongly positive for casein gene expression, an early differentiation marker in mammary secretory differentiation, but are negative for WAP, a protein expressed during terminal differentiation of the lactogenic phenotype. Similarly, WAP/Int3 mammary dysplasias are positive for casein expression and negative for WAP in postpartum females. In contrast MMTV-LTR/Int3 mammary tumors are negative for both of these markers specific for the lactational phenotype. This indicates that the cells giving rise to WAP/Int3 mammary tumors originate from the secretory epithelial cell progenitors whereas the MMTV-LTR/Int3 tumors appear early and arise from the ductal progenitor cells or from the individual mammary epithelial stem cells. Evidence for the latter conclusion comes from the observation that MMTV-LTR/Int3 glands are not able to grow and fill epithelium-divested mammary fat pads when transplanted to normal virgin hosts, suggesting that the clonogenic mammary stem cells are non-functional in MMTV-LTR/Int3 mammary transplants. In contrast, WAP/Int3 mammary transplants are successful in reconstituting a full mammary ductal system in more than 88% of the normal gland-free mammary fat pads implanted.
