Transgenic Expression of the Transforming Growth Factor beta1 in Developing Secretory Mammary Epithelium (WAP-TGFb1)

Summary

Mice, bearing a transgene comprised of the genomic fragment containing the entire WAP gene including the whey acidic protein gene promoter and possessing a c-DNA sequence encoding the Porcine TGF-b1 protein inserted into its 1st exon with appropriate stop signals etc., fail to lactate. Lactational failure is due to the inability of WAP-TGFb1 pregnant females to develop and maintain secretory lobules sufficient to provide milk for the offspring. The paucity of lobular development is linked to an increased apoptotic index in the secretory epithelium of the trangenic females. Transplantation studies prove that the developmental defect is intrinsic to the transgenic epithelium and that the effects of WAP-TGFb expression are not paracrine but either autocrine or intracrine. Mammary ductal development appears to be completely normal in situ, however transplantation of transgenic gland into normal virgin hosts demonstrates a diminished ability of TGFb mammary epithelium to repopulate epithelium-free mammary fat pads. Therefore WAP-TGFb expression not only impairs lobular progenitor development but promotes an early senescence of the regenerative capacity of mammary ductal epithelium. From these observations it is proposed that mammary epithelial stem cells may produce two functionally distinct epithelial progenitors; one capable of producing daughters committed only to ductal formation, the other capable only of producing progeny committed to lobular function.

Citation
Jhappan,C., Geiser,A.G., Kordon,E.K., Bagheri,D., Hennighausen, L., Roberts,A.B., Smith,G.H., and Merlino,G. (1993) Targeting of a transforming growth factor b1 transgene to the pregnant mammary gland inhibits alveolar development and lactation. EMBO J. 12:1835-1845.

Kordon, E.,K., McKnight,R.A., Jhappan,J., Hennighausen,L., Merlino,G., and Smith,G.H. (1995) Ectopic TGFb1 expression in the secretory mammary epithelium induces early senescence of the epithelial stem cell population. Developmental Biol. 168:47-61.

Background
TGFb1,2,3 have previously been shown to inhibit ductal growth and extension when applied to developing postpubertal mouse mammary glands. Expression of TGFb1 from the MMTV-LTR promoter was reported to transiently inhibit ductal growth in transgenic mice but had no detectable effect on lobular development or function. However, targeting the TGFb1 expression to developing secretory epithelium with the WAP promoter demonstrates that this growth factor can exert a major effect on the survival of lobular epithelium, suggesting a role for TGFb1 in remodeling of the gland following the cessation of lactation. Transplantation of TGFb1 mammary gland to non-transgenic hosts demonstrates inability for lobular development and maintenace is property inherent to the transgenic epithelium.

Transgene
A porcine TGFB1 cDNA was inserted into the Asp718 site within the first exon of a 7kb mouse WAP gene.

mouse strain
The transgene was introduced into a FVB/N background and the phenotype has been validated in several different genetic backgrounds (Smith,G.H. unpublished).

Mammary Phenotype

ducts
Normal branching and complete development in situ in virgin females, no apparent deficiency in side branching during pregnancy. Overall ductal outgrowth during puberty and pregnancy appears to be normal.

alveoli
During the first 1/3 of pregnancy whole mounts of TGFb1 and normal glands are indistinguishable. Subsequent lobular development in the second and third trimester of pregnancy is severely diminished in TGFb1glands, resulting in appearance of early pregnancy. The persisting lobules are small but appear to function normally in terms of milk protein production.

Alveolar development is impaired and in none of the mice the fat pad is completely filled during pregnancy. There is variation in alveolar outgrowth; in some of the miceat parturition, the alveolar structures are almost absent, and in others they mimic an 18 day pregnant gland.

epithelial cell morphology
Increased incidence of apoptotic cells along ducts and in developing lobules(TGFb1), an increased frequency of mitotic figures are present among the lobular cells throughout 1st two trimesters of pregnancy(TGFb1) suggesting expression of transgene does not interfere with cell cycling in developing lobules.

gene expression
Transgene expression is restricted rather sharply to mammary tissue with large increase at mid-pregnancy through paturition.

key words
WAP,whey acidic protein, transgenic, transforming growth factor, TGFb1, mammary development, senescence, stem cells, transplantation, alveolar development, agalactia, deregulated gene expression

Contributed By

Lothar Hennighausen and Gil Smith
National Institutes of Health
Bldg. 10, Rm. 5B
Bethesda, MD 20892
Phone: (301) 496-2385
Fax: 301-496-0839
e-mail: (e-mail mammary@nih.gov)