Transgenic mice expressing a truncated Int3 gene product in mammary tissue (MMTV-LTR-Int3)
Summary
Note:
the truncated int3 has also been expressed under control of the WAP-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
Int3 is a gene that is commonly mutated in Mouse Mammary Tumor Virus (MMTV) induced mammary tumors in mice (~20% in fetal mice).
Int3 is closely related to the Notch (Drosophila) gene family, a family of genes involved in cell fate selection during organogenesis and embryogenesis. All Int3 mutations found in mammary tumors result from the insertion of an MMTV provirus within the portion of the gene encoding the transmembrane domain (a target of approx. 200 bp). The insertions occur in frame and result in a truncated, viral-promoter-driven, Int3 mRNA which encodes the entire cytoplasmic domain of the Int3 protein. This mutation results in in the expression of the "active" form of the Int3 protein unencumbered by its association with the extracellular regulatory portion. In transgenic mice expressing a genomic fragment consisting of the 3' MMTV-LTR juxtaposed to the portion of the Int3 gene encoding the cyctoplasmic domain, the result is impaired mammary gland development and subsequent tumorigenesis.
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
Poor ductal extension and development in normally cycling virgin females; ducts are induced to completely fill the mammary fat pads with the onset of pregnancy or the introduction of pellets (slow release) containing estrogen, progesterone and hydrocortisone. Numerous dysplastic and swollen ducts are apparent under the dissecting microscope.
alveoli
Essentially no lobular development occurs in pregnant MMTV-LTR-Int3 females and lactation is absent. Little or no milk protein synthesis or milk fat globule formation is present.
Gene expression
Expression of the transgene was found in epididymis, salicary gland, Harderian gland, facial mucosal glands and mammary gland. Each of these tissues developed dysplasia and developmental abnormalities.
Tumorigenesis
Multiple mammary tumors occur as early as 1.5 months of age (avg. 3.5 month) in both virgin and breeding females. Salivary hyperplasia is always present during the same period.
Transplantation studies
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. Expression of the intracellular region of Int3 from the MMTV-LTR promoter in MMTV-LTR/Int3 transgenic mice (FVB3) blocks normal ductal branching morphogenesis in postpubertal females. However, in both models secretory lobular development is severely depressed during pregnancy resulting in a non-lactational phenotype. The normal development of ducts in WAP/Int3 mice suggests that the transgene is not expressed in the non-secretory ductal epithelial progenitor cells. A similar conclusion resulted from studies on the effects of TGFb1 on mammary lobular development in WAP/TGFb1 transgenic mice (information) and from the absence of any effect on mammary ductal growth and development in a variety of WAP promoter driven transgenic mouse models. In agreement with the restriction of WAP expression to mammary secretory epithelial progenitors, the mammary ducts ofWAP/Int3 males remain tumor-free in contrast to those in MMTV LTR/Int3 males information). WAP/Int3 and MMTV-LTR/Int3 transgenic mouse lines are therefore appropriate for distinguishing the effects of Int3 on different epithelial cell subsets in the differentiating mammary gland.
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.
Mechanistic Implications
Transgene
A genomic fragment comprised of a portion of the Env gene and 3' MMTV-LTR of the inserted provirus contiguous and in frame with the entire Int3 gene encoding the cytoplasmic domain of the protein. Transcription was uinder the control of the MMTV-LTR.
mouse strain
The transgene was introduced into a FVB/N inbred background.
key words
Int3, Notch, cell fate, mammary gland, differentiation, neoplastic transformation, tumorigenesis, insertional mutation, mammary development, alveolar development, ductal development,
The founding fathers and mother of the MMTV-Int3 mouse
For a better view click on the picture
Contributed by
Gilbert H. Smith
National cancer Institute
National Institutes of Health
Bethesda, MD 20892
tel. (301) 496-2385
FAX (301) 402-0711
e-mail: smithg@ltiblp.nci.nih.gov
contributed: May 1996
last update: June 1998
