Probes for CRE

Forkhead box A2 (FOXA2) is a pioneer transcription factor involved in organ development, function, and cancer. In the uterus, FOXA2 is essential for pregnancy and expressed specifically in the glands of the endometrium. Loss of FOXA2 function occurs during development of endometrial cancer in humans. The current study describes the development of a mouse model for conditional expression of mouse FOXA2. Using a system consisting of a minigene located at the Rosa26 locus, we generated a CAG-S-mFOXA2 allele in embryonic stem cells and subsequently in mice; before activation, the minigene is silent because of a floxed stop cassette inserted between the promoter and the transgene. To validate functionality, mice with the CAG-S-mFOXA2 allele were crossed with progesterone receptor (Pgr)-Cre mice and lactotransferrin (Ltf)-iCre mice that express Cre in the immature and adult uterus, respectively. In immature Pgr-Cre-CAG-S-mFoxa2 mice, FOXA2 protein was expressed in the luminal epithelium (LE), glandular epithelium (GE), stroma, and inner layer of the myometrium. Interestingly, FOXA2 protein was not observed in most of the LE of uteri from adult Pgr-Cre-CAG-S-mFoxa2 mice, although FOXA2 was maintained in the stroma, GE, and myometrium. The adult Pgr-Cre-CAG-S-mFoxa2 females were completely infertile. In contrast, Ltf-iCre-CAG-S-mFoxa2 mice were fertile with no detectable histological differences in the uterus. The adult uterus of Pgr-Cre-CAG-S-mFoxa2 mice was smaller, contained few endometrial glands, and displayed areas of partially stratified LE and GE. This transgenic mouse line is a valuable resource to elucidating and exploring FOXA2 function.

Abstract

BACKGROUND:

Transforming growth factor beta (TGFB) superfamily signaling is implicated in the development of sex cord-stromal tumors, a category of poorly defined gonadal tumors. The aim of this study was to determine potential effects of dysregulated TGFB signaling in the ovary using Cre recombinase driven by growth differentiation factor 9 (Gdf9) promoter known to be expressed in oocytes.

METHODS:

A mouse model containing constitutively active TGFBR1 (TGFBR1CA) using Gdf9-iCre (termed TGFBR1-CAG9Cre) was generated. Hematoxylin and eosin (H & E) staining, follicle counting, and immunohistochemistry and immunofluorescence analyses using antibodies directed to Ki67, forkhead box L2 (FOXL2), forkhead box O1 (FOXO1), inhibin alpha (INHA), and SRY (sex determining region Y)-box 9 were performed to determine the characteristics of the TGFBR1-CAG9Cre ovary. Terminal deoxynucleotidyl transferase (TdT) labeling of 3'-OH ends of DNA fragments, real-time PCR, and western blotting were used to examine apoptosis, select gene expression, and TGFBR1 activation. RNAscope in situ hybridization was used to localize the expression of GLI-Kruppel family member GLI1 (Gli1) in ovarian tumortissues.

RESULTS:

TGFBR1-CAG9Cre females were sterile. Sustained activation of TGFBR1 led to altered granulosa cell proliferation evidenced by high expression of Ki67. At an early age, these mice demonstrated follicular defects and development of ovarian granulosa cell tumors, which were immunoreactive for granulosa cell markers including FOXL2, FOXO1, and INHA. Further histochemical and molecular analyses provided evidence of overactivation of TGFBR1 in the granulosa cell compartment during ovarian pathogenesis in TGFBR1-CAG9Cre mice, along with upregulation of Gli1 and Gli2 and downregulation of Tgfbr3 in ovarian tumor tissues.

CONCLUSIONS:

These results reinforce the role of constitutively active TGFBR1 in promoting ovarian tumorigenesis in mice. The mouse model created in this study may be further exploited to define the cellular and molecular mechanisms of TGFB/activin downstream signaling in granulosa cell tumor development. Future studies are needed to test whether activation of TGFB/activin signaling contributes to the development of human granulosa cell tumors.