A novel LacZ reporter mouse reveals complex regulation of the progesterone receptor promoter during mammary gland development

To further our understanding of progesterone (P) as an endocrine mammogen, a PR(lacz) knockin mouse was generated in which the endogenous progesterone receptor (PR) promoter directly regulated lacZ reporter expression. The PR(lacz) mouse revealed PR promoter activity was restricted to the epithelial compartment during the prenatal and postnatal stages of mammary gland development. At puberty, PR promoter activity was unexpectedly robust and restricted to the body cells within the terminal end buds and to the luminal epithelial cells in the subtending ducts. In the adult, the preferential localization of PR(lacz) positive cells to the distal regions of ductal side branches provided a cellular context to the recognized mandatory role of P in ductal side-branching, and segregation of these cells from cells that undergo proliferation supported an intraepithelial paracrine mode of action for P in branching morphogenesis. Toward the end of pregnancy, the PR(lacz) mouse disclosed a progressive attenuation in PR promoter activity, supporting the postulate that the preparturient removal of the proliferative signal of P is a prerequisite for the emergence of a functional lactating mammary gland. The data suggest that PR expression before pregnancy is to ensure the specification and spatial organization of ductal and alveolar progenitor cell lineages, whereas abrogation of PR expression before lactation is required to enable terminal differentiation of the mammary gland.     

Transgenic expression of Cre recombinase from the tyrosine hydroxylase locus

Catecholaminergic neurons are affected in several neurological and psychiatric diseases. Tyrosine hydroxylase (TH) is the first, rate-limiting enzyme in catecholamine synthesis. We report a knockin mouse expressing Cre-recombinase from the 3'-untranslated region of the endogenous Th gene by means of an internal ribosomal entry sequence (IRES). The resulting Cre expression matches the normal pattern of TH expression, while the pattern and level of TH are not altered in the knockin mouse. Crossings with two different LacZ reporter mice demonstrated Cre-mediated genomic recombination in TH expressing tissues. In addition, LacZ was found in some unexpected cell populations (including oocytes), indicating recombination due to transient developmental TH expression. Our novel knockin mouse can be used for generation of tissue-specific or general knockouts (depending on scheme of crossing) in mice carrying genes flanked by loxP sites. This knockin mouse can also be used for tracing cell lineages expressing TH during development.     

Progesterone involvement in breast development and tumorigenesis--as revealed by progesterone receptor "knockout" and "knockin" mouse models

In light of recent clinical trials, the debate concerning the risks and benefits of progestin-based postmenopausal hormone replacement therapy (HRT) has reached a renewed level of urgency. Irrespective of the position taken, the consensus is that more basic research needs to be performed to address progesterone's fundamental role in mammary development and tumorigenesis. Towards this end, the progesterone receptor knockout (PRKO) mouse demonstrated that progesterone is essential for pregnancy-associated mammary gland ductal side-branching and alveologenesis and that these morphological changes are dependent on progesterone-induced mammary epithelial proliferation. Importantly, the PRKO mouse showed that the progesterone-proliferative signal significantly contributes to mammary tumor susceptibility in an established mammary tumor model. Insight into the cellular mechanism(s) by which progesterone affects mammary morphogenesis has been disclosed by a new PR-LacZ knockin mouse, which revealed that PR's spatial expression pattern undergoes precise choreographed distributional changes that precede key stages in postnatal mammary development. In the case of early pregnancy, the segregation of cells undergoing progesterone-induced proliferation from those that express PR implicates a paracrine mode of action for progesterone-induced mammary epithelial proliferation, whereas the preparturient decline of PR expression underscores the need to remove this signal for full functional differentiation of this tissue. Our findings support the proposal that the mammary gland's normal response to the progesterone-signal is dependent upon specific spatial organizational patterns of PR expression and that derailment in these cellular processes may contribute to abnormal mammary development, including cancer. This review concludes by emphasizing the need to identify the downstream molecular targets that mediate progesterone's effects in this tissue. Identification of such targets will not only enhance our mechanistic understanding of progesterone's role in mammary development and cancer, but may also facilitate the formulation of new design strategies in breast cancer diagnosis and/or treatment.     

Targeting reverse tetracycline-dependent transactivator to murine mammary epithelial cells that express the progesterone receptor

Through an established gene-targeting strategy, reverse tetracycline-dependent transactivator (rtTA) was targeted downstream of the murine progesterone receptor (PR) promoter. Mice were generated in which one (PR(+/rtTA)) or both (PR(rtTA/rtTA)) PR alleles harbor the rtTA insertion. The PR(+/rtTA) and PR(rtTA/rtTA) knockins exhibit phenotypes identical to the normal and the progesterone receptor knockout mouse, respectively. Crossed with the TZA reporter, which carries the TetO-LacZ responder transgene, the PR(+/rtTA)/TZA and PR(rtTA/rtTA)/TZA bigenics exhibit doxycycline-induced beta-galactosidase activity specifically in progesterone responsive target tissues such as the mammary gland, uterus, ovary, and pituitary gland. In the case of the PR(+/rtTA)/TZA mammary epithelium, dual immunofluorescence demonstrated that PR expression and doxycycline-induced beta-galactosidase activity colocalized; beta-galactosidase was not detected in the absence of doxycycline. Although both the PR(+/rtTA) and PR(rtTA/rtTA) knockins represent innovative animal models with which to further query progesterone's mechanism of action in vivo, the PR(rtTA/rtTA) mouse in particular promises to provide unique insight into the paracrine mechanism of action, which underpins progesterone's involvement in mammary morphogenesis with obvious implications for extending our understanding of this steroid's role in breast cancer progression.     

A mouse model to dissect progesterone signaling in the female reproductive tract and mammary gland

Considering the regulatory complexities of progesterone receptor (PR) action throughout the female reproductive axis and mammary gland, we generated a mouse model that enables conditional ablation of PR function in a spatiotemporal specific manner. Exon 2 of the murine PR gene was floxed to generate a conditional PR allele (PR^flox) in mice. Crossing the PR^flox/flox mouse with the ZP3‐cre transgenic demonstrated that the PR^flox allele recombines to a PR null allele (PR^d). Mice homozygous for the recombined null PR allele (PR^d/d) exhibit uterine, ovarian, and mammary gland defects that phenocopy those of our previously described PR knockout (PRKO) model. Therefore, this conditional mouse model for PR ablation represents an invaluable resource with which to further define in a developmental and/or reproductive stage‐specific manner the individual and integrative roles of distinct PR populations resident in multiple progesterone‐responsive target sites. genesis 48:106–113, 2010. © 2009 Wiley‐Liss, Inc.     

Murine progesterone receptor expression in proliferating mammary epithelial cells during normal pubertal development and adult estrous cycle. Association with eralpha and erbeta status.

The ovarian steroids estrogen and progesterone are important in directing the normal growth and development of the mouse mammary gland. Previously, we have demonstrated that the majority of proliferating mammary epithelial cells do not express estrogen receptor-alpha (ERalpha). In this study we examined the relationship between progesterone receptor (PR) expression and proliferation in mammary epithelial cells using simultaneous immunohistochemistry for progesterone receptor (PR) and tritiated thymidine [(3)H]-Tdr) autoradiography. Results showed that the majority (>80%) of mammary epithelial cells labeled with [(3)H]-Tdr were PR-positive in the terminal end buds (TEBs) of pubertal mice and the ducts of pubertal and adult mice. Whereas the majority of mammary epithelial cells were also PR-positive, the basal cell population, which comprises the minority of mammary epithelial cells in the mammary ducts, was predominantly PR-negative. Nevertheless, the PR-positive phenotype remained the major proliferating cell type in the basal population. These findings suggest that the progesterone signaling pathway is involved in the proliferation of basal cell populations, potentially directing formation of tertiary side branching during pubertal development and alveolar bud formation in adult glands. A proportion of the basal cells exhibited weak expression of ERbeta, suggesting that the role of ERbeta in mediating normal estrogen-induced responses should be further studied. (J Histochem Cytochem: 47:1323-1330, 1999)     

A mouse model engineered to conditionally express the progesterone receptor‐B isoform

Using a Rosa26 gene targeting strategy in mouse embryonic stem cells, we have generated a new transgenic mouse (Pgr‐B ^LSL), which is designed to conditionally express the epitope‐tagged mouse progesterone receptor‐B (PGR‐B) isoform when crossed with a specific cre driver mouse. To functionally validate this transgenic mouse, we crossed the Pgr‐B ^LSL mouse with the MMTV‐CREA transgenic mouse to create the MMTV‐CREA/Pgr‐B ^LSL bigenic (termed PR‐B:OE to denote PGR‐B o vere xpressor). As expected, transgene‐derived PGR‐B protein was specifically targeted to the virgin mammary gland epithelium. At a functional level, the PR‐B:OE bigenic exhibited abnormal mammary morphogenesis—dilated epithelial ducts, precocious alveologenesis and lateral side‐branching, along with a prominent proliferative signature—that resulted in pregnant PR‐B:OE mice unable to exhibit mammary gland terminal differentiation at parturition. Because of this developmental failure, the PR‐B:OE mammary gland was incapable of producing milk resulting in early neonatal death of otherwise healthy litters. This first line of analysis demonstrates the utility of the Pgr‐B ^LSL mouse to examine the role of the PGR‐B isoform in different physiologic and pathophysiologic systems that are responsive to progesterone.     

Cell-Specific Cre Recombinase Expression Allows Selective Ablation of Glutamate Receptors from Mouse Horizontal Cells

In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing.     

Z/EG, a double reporter mouse line that expresses enhanced green fluorescent protein upon Cre-mediated excision

The Cre/loxP system has become an important tool in designing postintegrational switch mechanisms for transgenes in mice. The power and spectrum of application of this system depends on transgenic mouse lines that provide Cre recombinase activity with a defined cell type-, tissue-, or developmental stage-specificity. We have developed a novel mouse line that acts as a Cre reporter. The mice, designated Z/EG (lacZ/EGFP), express lacZ throughout embryonic development and adult stages. Cre excision, however, removes the lacZ gene, which activates expression of the second reporter, enhanced green fluorescent protein. We have found that the double-reporter Z/EG line is able to indicate the occurrence of Cre excision from early embryonic to adult lineages. The advantage of the Z/EG line is that Cre-mediated excision can be monitored in live samples and that live cells with Cre-mediated excision can be isolated using a single-step FACS. It will be a valuable reagent for the increasing number of investigators taking advantage of the powerful tools provided by the Cre/loxP site-specific recombinase system.     

Transgenic Cre expression mice for generation of erythroid-specific gene alterations

Transgenic mice that express Cre recombinase in erythroid cell lineages were developed so that genes affecting erythropoiesis/hematopoiesis may be altered without necessarily affecting fetus viability. A micro-LCR cassette-beta-globin promoter-Cre recombinase gene (microLCR-betapr-Cre) construct was synthesized and used to generate transgenic mice. Concurrently, we produced mice containing a microLCR-loxP-flanked beta sickle gene (microLCR-loxP-beta(S)-loxP) construct. microLCR-betapr-Cre mice with intact transgenes in variable copy number were identified. Cre expression was assessed by RNAse protection and RT-PCR. Cre function was ascertained by breeding to microLCR-loxP-beta(S)-loxP mice. We demonstrate that beta(S) expression was not detected in the blood of bigenics, but the gene was present in nonerythroid cells. Thus, excision of the loxP-flanked beta(S) gene was restricted to erythroid cell lineages.     

Efficient gene-specific expression of cre recombinase in the mouse embryo by targeted insertion of a novel IRES-Cre cassette into endogenous loci.

Site specific recombinases have provided the experimental strategy necessary to modulate the expression of gene products in the mouse embryo. In this study we have exploited Cre recombinase to develop a widely applicable cell marking system which functions efficiently even at early post-implantation embryonic stages. Importantly, the techniques and reagents derived in this study are generally applicable to any recombinase driven approach, including strategies to temporally and spatially modulate endogenous or ectopic gene expression in the embryo. The cell marking scheme has two essential components which were derived as separate mouse lines. The first line carries a universal conditional lacZ reporter (UCR) locus which was prepared by using gene targeting in a novel approach to modify a ubiquitously expressed retroviral lacZ promoter trap insertion. The UCR locus is silent until it undergoes a Cre mediated DNA rearrangement to restore lacZ expression. To generate the Cre expressing allele, we outline a flexible strategy which requires the introduction of a novel IRES-Cre cassette into exon sequence of an endogenous locus by gene targeting. We successfully demonstrate this approach by generating a Cre expressing allele of the EphA2 gene, an Eph receptor protein tyrosine kinase expressed early in development. Analysis of double heterozygote embryos clearly demonstrates that Cre recombinase is expressed in vivo from the EphA2 IRES-Cre allele, and that the conditional reporter locus is efficiently restored in EphA2-expressing cells as early as 7.5 dpc. This cell marking experiment establishes the feasibility of expressing Cre recombinase from a single copy allele in the embryo and demonstrates the utility of the conditional reporter mouse which can be used in the analysis of any Cre expressing allele.