Several Wnt genes are expressed in the postnatal mouse mammary gland and are thought to be involved in mammary gland development. Ectopic expression of Wnt-1, which is not normally expressed in the mammary gland, drives the formation of a pre-neoplastic hyperplasia. Cell culture-based assays have shown that Wnt-1 and some mammary-expressed Wnts transform C57MG cells. This has led to the suggestion that Wnt-1 functions as an oncogene through the inappropriate activation of developmental events that are normally controlled by the ‘transforming’ class of Wnts. In this study, Wnt-7b was expressed in vivo using recombinant retroviruses. Wnt-7b did not alter normal mammary gland development despite having similar effects to Wnt-1 in cell culture. We conclude that the in vitro classification of Wnts as ‘transforming’ does not correlate with the transformation in vivo. To facilitate the analysis of Wnt-expression, a lacZ-containing, bicistronic recombinant retrovirus was developed. Immunohistochemistry and electron microscopy identified retrovirally transduced myoepithelial and luminal epithelial cells in normal and hyperplastic tissues. The distribution of transduced cells in mammary outgrowths was consistent with current models of mammary stem cell identity.

The Wnt genes encode a large family of secreted glycoproteins that are conserved in a range of species (reviewed in Nusse and Varmus, 1992). The prototype member, Wnt-1, was isolated as a common integration site for mouse mammary tumour virus in mammary tumours (Nusse and Varmus, 1982) and was later shown to be homologous to the Drosophila developmental control gene wingless (Nusse et al., 1991). The spatial and temporal expression patterns of Wnt genes suggest that they are involved in a range of developmental processes (Moon et al., 1997). Targeted disruption in mice has demonstrated functional roles for Wnt-1, Wnt-2, Wnt-3, Wnt-3a, Wnt-4, Wnt-5A and Wnt-7A in processes including gastrulation, midbrain patterning, limb and kidney development (Liu et al., 1999a; Monkley et al., 1996; Stark et al., 1994; Takada et al., 1994; Thomas et al., 1991; Yamaguchi et al., 1999) (for an overview see the Nusse homepage at http://www.stanford.edu/approx. rnusse/wntwindow.html). Many additional roles for Wnt ligands have been suggested on the basis of tissue distribution and ectopic expression analyses. Wnt proteins associate with the cell surface and extracellular matrix (Papkoff and Schryver, 1990; Ramakrishna and Brown, 1993; Reichsman et al., 1996; Schryver et al., 1996). In vivo, the Wingless protein of Drosophila tightly localises within a few cell diameter lengths of its site of expression (Gonzalez et al., 1991), suggesting that members of the Wnt gene family function in short-range cell-cell signalling.

Wnt ligands bind to receptors of the frizzled family at the cell surface (Orsulic and Peifer, 1996). The Wnt signal is transduced through a series of intracellular molecules leading to the stabilisation of β-catenin, which then enters the nucleus and activates the transcription of target genes as part of a complex with T-Cell Factor (TCF) DNA binding proteins (reviewed in Dale, 1998; Wodarz and Nusse, 1998). At least 16 Wnt genes and 7 Frizzled genes have been identified in mammalian genomes and recent studies have shown that particular Wnt-Frizzled combinations activate either the β-catenin/TCF pathway or non-canonical pathways involving proteins such as PKC, PI3Kinase and Jun kinase (JNK) (Boutros et al., 1998; Cook et al., 1996; Li et al., 1999; Liu et al., 1999b; Moriguchi et al., 1999; Sheldahl et al., 1999; Slusarski et al., 1997; Zhang et al., 1999). Predicting the activation of Wnt signal transduction in particular tissues is further complicated by the discovery of families of secreted Wnt inhibitors that bind and interfere with Wnt function (Finch et al., 1997; Hsieh et al., 1999; Piccolo et al., 1999; Wang et al., 1997).

Inappropriate activation of components of the canonical Wnt signalling pathway has been implicated in a number of different cancers. In colon cancer, most tumours have activated the pathway either through the loss of APC (Adenomatous Polyposis Coli protein) function or the mutation of β-catenin (reviewed in Polakis, 1999). N-terminal mutation of β-catenin has also been detected in hepatocellular carcinomas and melanomas. A range of indirect evidence implicates deregulated Wnt signalling in a variety of other tumour types (reviewed in Smalley and Dale, 1999). The importance of the oncogenic deregulation of the pathway is starting to be understood as cyclin D1 and myc have been shown to be transcriptional targets of TCF factors (He et al., 1998; Shtutman et al., 1999).

Expression of Wnt-1 in the mammary gland of virgin mice has been shown to induce a pregnancy-like hyperplasia and cause occasional tumours (Edwards et al., 1992; Lin et al., 1992; Tsukamoto et al., 1988). Most reports have shown that Wnt-1 is not normally expressed in the mammary gland. The capacity of the gland to respond to the Wnt-1 signal has led to the hypothesis that Wnt-1 drives tumourigenesis by activating proliferative responses which are under the control of Wnts that are normally expressed in the mammary gland. At least six Wnt genes are normally expressed in the mouse mammary gland and alterations in Wnt expression correlate with developmental changes (Buhler et al., 1993; Gavin and McMahon, 1992; Weber-Hall et al., 1994). In particular, Wnt-4 expression is strongly induced during pregnancy and ectopic expression of Wnt-4 in vivo induces a pregnancy-like growth pattern in reconstituted glands of virgin mice (Bradbury et al., 1995).

To look at the effect of inappropriate expression of a variety of genes on breast development, we have utilised a transplantation system in the mouse mammary gland (Edwards et al., 1988). Genes are introduced into primary mouse mammary epithelial cells in culture using helper-free retroviral vectors. The altered cells are transplanted into the mammary fat pad of young mice from which all the endogenous epithelium has been surgically removed. The transplanted epithelium repopulates the fat pad and the resultant epithelial tree can be examined for alterations in development induced by the introduced gene.

In this paper, we describe a further development of the mammary transplantation system to allow the analysis of subtle cellular changes by developing retroviruses that allow the histochemical localisation of transduced cells by whole-mount analysis, light and electron microscopy. We show that Wnt-1 and Wnt-7b, despite having similar effects on mammary epithelial cells in culture, have markedly different effects when expressed in vivo.

Routine cell culture and harvest of primary mouse mammary epithelial cells

The ecotropic packaging cell line GP+E-86 (Markowitz et al., 1988) and NIH 3T3 fibroblasts (ATCC CRL-1658) were grown in Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Life Technologies, Paisley, UK) supplemented with 10% foetal bovine serum (FBS; Gibco). 10T1/2 fibroblasts (ATCC CCL-226) were grown in DMEM supplemented with 5% FBS. C57MG cells were grown in DMEM with 10% FBS and 10 μg ml−1 insulin (Brown et al., 1986; Sigma, Poole, Dorset, UK).

Primary mouse mammary epithelial cells (MMECs) were harvested from 10-12 week old virgin female Balb/C mice essentially as described (Smalley et al., 1998), with minor variations. These were the use of 3 mg ml−1 collagenase and 1.5 mg ml−1 porcine trypsin (Sigma) in serum-free Leibowitz L15 medium (Gibco) for the fat-pad digestion as well as the culture of the harvested primary MMECs in 1:1 DMEM: Ham’s F12 (Gibco) supplemented with 10% FBS, 10 μg ml−1 insulin, 10 ng ml−1 EGF and 10 ng ml−1 cholera toxin (all additives from Sigma) in a 5% CO2/air atmosphere. Low oxygen conditions as previously described for clonal culture of MMECs (Smalley et al., 1998) are not required for bulk culture of MMECs. The cells were cultured unsorted, as mixed epithelial populations.

Construct details

The pINA retroviruses have previously been described (Bradbury et al., 1994). To generate bicistronic retroviral constructs, Wnt-1, -5b and -7b cDNAs (originally obtained from Dr A. McMahon) were tagged at the carboxy terminus with the ‘myc’ epitope by PCR strategies adding the amino acids EQKLISEEDL, recognised by the monoclonal antibody 9E10. Myc-tagged Wnt-1, -5b and -7b constructs were cloned into the retroviral vector ‘polyPOZ’. polyPOZ was derived from pLNPOZ (a kind gift from A. D. Miller, Fred Hutchinson Cancer Center) by the substitution of the neomycin coding sequence with a polylinker. pLNPOZ contains the Internal Ribosome Entry Site (IRES) from poliovirus 5′ of the β-galactosidase (β-gal) coding sequence, allowing its internal translation initiation (Adam et al., 1991). To generate polyPOZ from pLNPOZ, the neomycin-resistance coding sequence was removed by a BclI partial digest followed by an XhoI digest to completion. A polylinker comprising the sites BclI – NotI – BglII – StuI – HinDIII – SalI – XhoI was custom synthesised, annealed and cloned in at this point.

Preparation of polyPOZ retroviruses

Stable retroviral producer lines were generated by superinfection (Morgenstern and Land, 1991). Briefly, supernatants containing viral particles were collected from GP+E-86 cells transiently transfected with Wnt-polyPOZ or polyPOZ vector constructs. These were used to superinfect tunicamycin-treated GP+E-86 cells in the presence of 8 μg ml−1 polybrene (hexadimethrine bromide; Sigma) to make stable polyPOZ producer cells. High titre lines were generated by sequential fluorescence-activated cell sorting (FACS) on the basis of β-gal expression using the fluoresceinated substrate fluorescein di-β-D-galactoside (FDG) (MacGregor et al., 1991). A ‘Fluoreporter Lac Z’ flow cytometry kit (Molecular Probes, Leiden, Holland) was used according to the manufacturer’s protocol. The top 2-5% of cells with respect to β-gal activity were sorted, expanded then resorted until the resultant population all displayed high levels of β-gal activity, as determined by FACS analysis and visually confirmed by ‘X-gal’ staining (MacGregor et al., 1991).

Retroviral producer lines were expanded until 80% confluence then the medium was replaced with DMEM supplemented with 10% Nuserum (Collaborative Research, Universal Biologicals, London, UK). Retroviral supernatants were harvested 48 hours later, concentrated five-to tenfold by size exclusion ultrafiltration using the Amicon Stirrer Cell with a ZM500 membrane (Amicon, Gloucestershire, UK) and then passed through a 0.45 μm filter. Concentrated viral supernatants were divided into portions, snap frozen on dry ice and stored at −80°C. Assays for replication-competent virus were performed on all stocks using the two methods of marker transfer and reverse transcriptase assay (Cepko and Pear, 1995). All stocks were determined free of replication competent virus. Retroviral titres were determined on NIH 3T3 cells by serial dilution and consequent X-gal staining. Titres fell typically in the range 1×106-1×107lacZ transducing units ml−1 (LTU ml−1).

The C57MG transformation assay was carried out essentially as previously described for 10T1/2 cells (Bradbury et al., 1994).

Characterisation of Wnt-polyPOZ viruses

To assess the relationship between the numbers of LTU ml−1 in retroviral supernatants and the subsequent number of cells transduced in culture, 3T3 cells were plated at a density of 2×105 well−1 in six-well plates and transduced with Wnt-1myc-polyPOZ virus in the presence of 8 μg ml−1 polybrene. Transduction was at a multiplicity of infection (MOI; the ratio of LTU ml−1 viral supernatant to plated cells) of 4. After 3 days the number of transduced cells was analysed on the basis of β-gal activity using FDG and FACS analysis.

To confirm that cells transduced with Wnt-polyPOZ viruses did express both Wnt and β-galactosidase proteins, 3T3 cells were cultured on coverslips and transduced with polyPOZ retroviral vectors expressing Wnt-1myc or Wnt-7bmyc. Transduction was performed at 37°C for 3 hours in the presence of 8 μg ml−1 polybrene, after which the cells were re-fed with growth medium and incubated for 48 hours. They were then fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) and permeabilised for 10 minutes in 0.2% Triton X-100 in PBS. The coverslips were stained with antibodies 9E10 (Sigma) to the myc epitope and Z3781 (Promega) to β-galactosidase using standard indirect immunofluorescence techniques and examined under both transmitted and epifluorescent illumination.

The CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay was used to measure 10T1/2 fibroblast proliferation (Promega). Cells were seeded at a range of cell densities in a 96-well plate (4×103-4×104 cells cm−2), incubated overnight, transduced at an MOI of approximately 5 and then cultured for a further 8 days before analysis. Transduction was performed at 37°C for 2 hours in the presence of 8 μg ml−1 polybrene.

Retroviral transduction and transplantation of primary mouse mammary epithelial cells

Primary MMECs were cultured for 1 week, during which time they were subjected to four rounds of retroviral transduction. Typically, medium was aspirated from the cells and 4-6 ml of retroviral supernatant plus 8 μg ml−1 polybrene was added to each flask of cells. After 2 hours at 37°C the cultures were re-fed with fresh growth medium. Retroviral transduction was carried out when the cells were subconfluent, in order to maximise proliferation and thus retroviral integration. If necessary, the cells were passaged once in order to achieve this.

After 1 week, retroviral-transduced MMECs were harvested and transplanted into the fourth mammary fat pads of 3-week old syngeneic female Balb/C mice which had been cleared of endogenous epithelium as described (Bradbury et al., 1991). Between 5×105 and 1×106 cells in 10 μl serum-free L15 medium were transplanted into each fat pad. In general, one fat pad of each animal was transplanted with cells transduced with one of the Wnt/β-gal viruses, whilst the contra-lateral fat pad was transplanted with cells transduced with the control, polyPOZ vector resulting in expression of the β-galactosidase marker only in transduced cells.

Cells that were left over after transplantation were put back into culture for 2-3 days and then fixed and stained in one of three ways: with X-gal, to determine the approximate percentage of transplanted cells that had been transduced; with X-gal and an antibody (ICR2; from the Institute of Cancer Research Hybridoma Unit) against the epithelial cell surface marker Epithelial Membrane Antigen (EMA), to determine if the transduced cells were epithelial; and with antibodies against cytokeratins 14 and 18 (LE61 and LLOO2 respectively, kindly provided by Professor E. B. Lane, Department of Biochemistry, University of Dundee, UK), which enabled the cultured epithelial cells to be characterised as either luminal epithelial or myoepithelial in origin (Smalley et al., 1998).

The cells were given 8-10 weeks to proliferate through the cleared mammary fat pads, after which the fat pads were harvested and analysed. All animal work was carried out according to Home Office guidelines.

Analysis of reconstituted mammary fat pads

Mammary fat pads to be analysed were stretched out on glass slides and then fixed in 1% paraformaldehyde, 0.2% glutaraldehyde, 2 mM MgCl2, 5 mM EGTA, 0.02% NP40 in PBS for 4 hours at room temperature. They were then washed three times in PBS, 0.02% NP40 for 30 minutes each wash and then detached from the slides to ensure penetration of the X-gal stain (Whiting et al., 1991). Glands were left in the staining solution overnight at room temperature. The stain consisted of 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 2 mM MgCl2, 0.01% sodium deoxycholate, 0.02% NP40, 1 mg ml−1 X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside; Sigma). Following staining, glands were rinsed twice in PBS, 0.02% NP40, post-fixed for 4 hours in 4% paraformaldehyde in PBS and then dehydrated through a graded series of alcohols. The glands were then cleared in methyl salicylate for viewing. Following viewing, glands were either stored in 100% ethanol, processed for light and electron microscopy or carmine stained (Banerjee et al., 1976). For staining in carmine, glands were rehydrated to 70% ethanol, rinsed in distilled water and stained overnight in 0.2% carmine, 0.5% potassium aluminium sulphate in distilled water. Glands were then dehydrated, cleared in methyl salicylate and examined.

Light and electron microscopy

Small pieces of β-gal positive areas were dissected out under a low-power dissecting microscope, transferred to fresh 100% ethanol, rinsed twice in 100% ethanol and stored overnight at 4°C in 100% ethanol. Next day the pieces were transferred into a 50:50 mixture of ethanol:Lowicryl K4M (Agar Scientific, Stansted, Essex) at 0°C in a Leica automatic freeze substitution apparatus and allowed to infiltrate for 4 hours. The pieces were then transferred on to 0.5 ml of previously polymerised resin, overlaid with 1.5 ml of K4M and polymerised overnight at 0°C. For light microscopy immunolabelling, 0.5 μm sections were cut onto coated slides (1% formalised gelatin) and incubated overnight in anti-smooth muscle actin (Sigma) diluted 1:4000, followed by goat-anti-mouse 5 nm gold (GAM5) and silver enhanced for 24 minutes using IntenSE enhancement kit (Amersham). Slides were photographed unstained by transmitted light to show β-gal product (blue-green) and smooth muscle actin (SMA)-containing cells (black). The slides were then stained with dilute Toluidine Blue to show cell morphology and the same areas rephotographed with both transmitted and epi-polarised light (under which the SMA is visible as a silver-blue stain). For electron microscopy, 80 nm sections were cut on to Formvar-coated gold grids, stained with uranyl acetate and viewed in a Philips CM10 Biotwin at 60 kV accelerating voltage. Immuno-electron microscopy for SMA was as previously described (Robertson et al., 1992).

Transduction and selection of C57MG mammary epithelial cells with neomycin-resistance/Wnt-expressing pINA retroviral vectors generated colonies with cell morphologies and densities that were consistent with the previous classification of Wnts as ‘transforming’ and ‘non-transforming’. In this classification, Wnt-1 and Wnt-7b transformed C57MG cells while Wnt-4 and Wnt-5b failed to transform (Fig. 1; Wong et al., 1994). The relevance of the C57MG assay for normal mammary epithelial transformation was brought into question by our previous studies that showed that Wnt-4 induced a mammary epithelial hyperplasia despite its failure to transform C57MG cells (Bradbury et al., 1995).

Fig. 1.

C57MG clone morphology following transduction with Wnt-expressing retroviruses. C57MG cells were transduced with recombinant pINA retroviruses and selected for neomycin resistance. Giemsa staining showed even staining of control INA (C) transduced colonies. Wnt-1-INA (1) and Wnt-7b-INA (7b) colonies appear darker due to the higher density of the transformed cells. Wnt-5b-INA (5b), and Wnt-4-INA (4) have little or no effect.

Fig. 1.

C57MG clone morphology following transduction with Wnt-expressing retroviruses. C57MG cells were transduced with recombinant pINA retroviruses and selected for neomycin resistance. Giemsa staining showed even staining of control INA (C) transduced colonies. Wnt-1-INA (1) and Wnt-7b-INA (7b) colonies appear darker due to the higher density of the transformed cells. Wnt-5b-INA (5b), and Wnt-4-INA (4) have little or no effect.

We previously attempted to express Wnt-7b in vivo and saw no effect on the epithelium, but these experiments were inconclusive due to our inability to show that the retrovirus was expressed in vivo. To overcome these problems we developed a novel retroviral vector, ‘polyPOZ’, derived from pLNPOZ (Abram et al., 1997; Adam et al., 1991; Ghattas et al., 1991). PolyPOZ expresses the gene of interest and β-galactosidase from a bicistronic vector containing an internal ribosome entry site (Fig. 2A; IRES). High-titer retroviral producer lines were generated by superinfection of GPE+E-86 producer cells with supernatant from transfected cells, followed by FACS sorting of the top 2-5% of expressing cells. Expressing cells were resorted for β-galactosidase expression until the resultant population displayed high levels of β-gal activity, as determined by FACS analysis (Fig. 2Bi). Retroviral titers were normally in the range 1×106-1×107 LTU per ml as measured by X-gal staining, while FACS sorting showed that 50% of all NIH3T3 cells could be made to express β-galactosidase at an MOI of 4 (Fig. 2Bii).

Fig. 2.

Generation of high-titer polyPOZ retroviruses. (A) Map of the polyPOZ retrovirus showing the polylinker into which Wnt-1 cDNAs were cloned. Translation of LacZ is mediated by the internal ribosome entry site (IRES). LTR, long terminal repeat sequence; ψ, psi packaging sequence. (B) (i) Preparation of viral stocks. Use of FACS to prepare high-titer retroviral producer stocks. High-titer lines were generated by sequential FACS on the basis of β-gal expression using the fluoresceinated substrate fluorescein di-β-D-galactoside (FDG). The top 2-5% of cells with respect to β-gal activity were sorted, expanded and then resorted. The x axis is a log scale in arbitrary units measuring fluorescence intensity; the y axis is a linear arbitrary scale in which 10-20,000 single cells were measured. Trace 1, Viral producer cell line after superinfection; trace 2, expanded cells following first FACS sort; trace 3, expanded cells following second FACS sort. (ii) Transduction of NIH3T3 cells. The efficiency of transduction by polyPOZ virus was measured as described above following transduction at an MOI of 4. The percentage of cells expressing above the indicated fluorescence gate is displayed.

Fig. 2.

Generation of high-titer polyPOZ retroviruses. (A) Map of the polyPOZ retrovirus showing the polylinker into which Wnt-1 cDNAs were cloned. Translation of LacZ is mediated by the internal ribosome entry site (IRES). LTR, long terminal repeat sequence; ψ, psi packaging sequence. (B) (i) Preparation of viral stocks. Use of FACS to prepare high-titer retroviral producer stocks. High-titer lines were generated by sequential FACS on the basis of β-gal expression using the fluoresceinated substrate fluorescein di-β-D-galactoside (FDG). The top 2-5% of cells with respect to β-gal activity were sorted, expanded and then resorted. The x axis is a log scale in arbitrary units measuring fluorescence intensity; the y axis is a linear arbitrary scale in which 10-20,000 single cells were measured. Trace 1, Viral producer cell line after superinfection; trace 2, expanded cells following first FACS sort; trace 3, expanded cells following second FACS sort. (ii) Transduction of NIH3T3 cells. The efficiency of transduction by polyPOZ virus was measured as described above following transduction at an MOI of 4. The percentage of cells expressing above the indicated fluorescence gate is displayed.

Myc-epitope tagged cDNAs for Wnt-1, Wnt-5b and Wnt-7b were cloned into polyPOZ and used to generate viral supernatants. Northern analysis demonstrated that unit length retroviral messages contained sequences for both Wnt and lacZ within transduced pools of NIH3T3 cells (data not shown).

Staining of virally transduced NIH3T3 cells for β-galactosidase and myc-epitope expression showed that >90% of cells coexpressed both markers, suggesting that lacZ expression served as a good marker for Wnt expression (Fig. 3A). To analyse the functional behaviour of the recombinant retroviruses in vitro, we chose to use 10T1/2 fibroblasts since they respond well to Wnt expression, are less prone to spontaneous transformation than C57MG cells and may be less variable in their range of responses (Bradbury et al., 1994; Shimizu et al., 1997). Transduction with polyPOZ-Wnt-1myc and polyPOZ-Wnt-7b myc altered 10T1/2 fibroblast morphology and increased cell proliferation (Fig. 3B). In these assays, Wnt-1myc appeared to have a slightly greater effect than Wnt-7bmyc on cell proliferation (Fig. 3Bii). Transduction with polyPOZ-Wnt-5bmyc retroviruses had little effect. In accordance with previous observations, the extent of over-proliferation of the 10T1/2 cells varied with the initial plating density and time spent at confluence (Fig. 3Bii; Bradbury et al., 1994).

Fig. 3.

Characterisation of Wnt-myc expressing retroviruses. (A) Cotranslation of Wnt-myc and β-galactosidase. NIH3T3 cells were transduced with polyPOZ-Wnt-1myc retroviruses. Transduced cells were costained for expression of the myc epitope and β-galactosidase. (i) Phase contrast image, (ii) Myc staining, (iii) β-galactosidase staining. Control ‘no first antibody’ stainings showed little or no background (data not shown). Bar, 40 μm. (B) Transduction of 10T1/2 fibroblasts. (i) Cell morphology assay.

Fig. 3.

Characterisation of Wnt-myc expressing retroviruses. (A) Cotranslation of Wnt-myc and β-galactosidase. NIH3T3 cells were transduced with polyPOZ-Wnt-1myc retroviruses. Transduced cells were costained for expression of the myc epitope and β-galactosidase. (i) Phase contrast image, (ii) Myc staining, (iii) β-galactosidase staining. Control ‘no first antibody’ stainings showed little or no background (data not shown). Bar, 40 μm. (B) Transduction of 10T1/2 fibroblasts. (i) Cell morphology assay.

The polyPOZ-Wnt-1myc, Wnt-7bmyc Wnt-5bmyc and control retroviruses were used to transduce primary mouse epithelial cells in culture (Fig. 4A). The epithelial identity of the transduced cells was confirmed by colocalisation of X-Gal staining and the epithelial membrane antigen marker (EMA). Cytokeratin staining demonstrated that the majority of cells in the cultures were cytokeratin 18-positive and cytokeratin 14-negative (CK18+/CK14−) or CK18+/CK14+ and were thus predominantly luminal epithelial in origin (data not shown; Smalley et al., 1998). Following transduction, the cells were transplanted into mouse mammary fat pads from which the endogenous epithelium had been surgically removed. After 8 weeks, the transplanted fat pads were analysed by X-Gal staining combined with whole-mount analysis (Fig. 4B).

Fig. 4.

Transduction of mouse mammary epithelial cells. (A) Transduction of primary mammary epithelial cells. polyPOZ control virus transduction of primary mammary epithelial cell culture revealed with X-Gal (i). (ii) Transduction of luminal epithelial cells revealed by costaining for X-Gal and (iii) and Epithelial Membrane Antigen (EMA). Bar, 40 μm. (B) Reconstituted mammary glands. Representative, reconstituted glands containing mammary epithelial cells transduced with polyPOZ-Wnt-1myc (i, ii), polyPOZ-Wnt-5bmyc (iii, x), polyPOZ-Wnt-7bmyc (iv,v,ix) and polyPOZ control (vi-viii) retroviruses. Transplants shown in ix and x were from 11- and 10-day pregnant animals, respectively. A total of 22 polyPOZ-Wnt-1myc, 35 polyPOZ-Wnt-5bmyc, 40 polyPOZ-Wnt-7bmyc and 49 polyPOZ control transduced glands were analysed. Glands containing X-Gal staining material were detected at a rate of 80%. i and iii were counterstained with Carmine (red); i and iv contain lymph nodes. Bars, 600 μm.

Fig. 4.

Transduction of mouse mammary epithelial cells. (A) Transduction of primary mammary epithelial cells. polyPOZ control virus transduction of primary mammary epithelial cell culture revealed with X-Gal (i). (ii) Transduction of luminal epithelial cells revealed by costaining for X-Gal and (iii) and Epithelial Membrane Antigen (EMA). Bar, 40 μm. (B) Reconstituted mammary glands. Representative, reconstituted glands containing mammary epithelial cells transduced with polyPOZ-Wnt-1myc (i, ii), polyPOZ-Wnt-5bmyc (iii, x), polyPOZ-Wnt-7bmyc (iv,v,ix) and polyPOZ control (vi-viii) retroviruses. Transplants shown in ix and x were from 11- and 10-day pregnant animals, respectively. A total of 22 polyPOZ-Wnt-1myc, 35 polyPOZ-Wnt-5bmyc, 40 polyPOZ-Wnt-7bmyc and 49 polyPOZ control transduced glands were analysed. Glands containing X-Gal staining material were detected at a rate of 80%. i and iii were counterstained with Carmine (red); i and iv contain lymph nodes. Bars, 600 μm.

Transplants made from polyPOZ-Wnt-1myc transduced cells showed a characteristic Wnt-1 hyperplasia in all mature ducts that stained blue with X-Gal (Fig. 4Bi,ii). No hyperplasia was seen observed in mature ducts that were negative for lacZ expression (shown by Carmine counterstain in Fig. 4Bi), supporting previous observations that Wnt-1 operates over relatively short cellular distances (Gonzalez et al., 1991). Interestingly, the degree of excess branching induced by Wnt-1 correlated with the intensity of staining for X-Gal, suggesting a concentration dependence in the behaviour of mammary epithelial cells to Wnt-1 ligand. In 3 glands out of 22 in one series of Wnt-1 transplants, X-Gal staining was detected in a gradient from high expressing ductal end buds to the non-expressing, subtending ducts (data not shown). The presence of a gradient, as distinct from a boundary, suggested that both the expressing and non-expressing cells were derived from integrated retroviruses that were sensitive to the differentiation/ proliferation status of the epithelial cells (i.e. endbud versus ductal) and not from a boundary between infected and uninfected cells. The observation that polyPOZ-Wnt-1myc transduced end buds were structurally normal suggested that Wnt-1 could not alter with the behaviour of the highly proliferative end bud structures, but could interfere with the development of subtending ducts.

By contrast with Wnt-1, when transplants were made after transduction with the polyPOZ-Wnt-7bmyc or Wnt-5bmyc retroviruses, no morphological changes were observed in transplanted regions expressing β-galactosidase (Fig. 4Biii-v).

In these experiments, the level of expression from polyPOZ-Wnt-7bmyc and Wnt-5bmyc retroviruses was equivalent to that of Wnt-1, as assessed by the range of intensity of X-Gal staining. End buds expressing polyPOZ-Wnt-5bmyc and Wnt-7bmyc also had normal structures (Fig. 4Bv). Preliminary analyses of transduced glands taken from pregnant animals showed normal lobular development in regions expressing Wnt-7bmyc and Wnt-5bmyc (Fig. 4Bix,x), suggesting that neither Wnt interfered with the initial stages of pregnancy induced lobular development.

In the majority of control, polyPOZ-Wnt-5bmyc and Wnt-7bmyc transplanted glands, X-Gal staining localised to contiguous clusters of cells comprising a section of duct (Fig. 4Bvi); however, at a frequency of approximately 1 gland in 10, a second distinct ‘speckled’ staining pattern was observed (Fig. 4Bviii). Regions containing both speckled and continuous staining patterns were not detected. Within expressing regions, the cell-cell variability in expression was slight in comparison to the variability in intensity between glands, supporting the suggestion that large regions of the gland were clonally derived from single retrovirally transduced progenitor cells (Kordon and Smith, 1998). We cannot exclude the possibility, however, that non-expressing regions within the gland resulted from selective inactivation of retroviral expression.

To identify individual transduced cells, sections were cut from X-Gal stained glands (Fig. 5). X-Gal precipitate remained visible in the sections, allowing the correlation of ductal organisation with viral expression. Analysis of control, Wnt-5bmyc and Wnt-7bmyc expressing cells showed normal polarised luminal cells surrounded by basal myoepithelial cells (Fig. 5i,ii; data not shown). Analysis of the ‘speckled’ ducts showed that transduced cells were both basally and luminally located (Fig. 5i). To enhance the identification of cell types, X-Gal containing sections were stained for expression of smooth muscle actin, a myoepithelial marker (Fig. 5iii,iv; Sapino et al., 1990). Wnt-1myc expressing cells displayed a range of phenotypes from a relatively normal architecture to completely disorganised structures. In the majority of ducts the lumen was not clearly defined or was structurally flawed (Fig. 5iv, right hand solid blue epithelial structure). In many regions, smooth muscle actin-positive and -negative cells clustered in disorganised exclusive groups. In other regions normal luminal-myoepithelial cell interactions were maintained while the ducts became multilayered with a myo-luminal-luminal-myo orientation (Fig. 5iv, left hand blue epithelial structure). As noted in the whole-mount analysis, the degree of disorganisation correlated with the intensity of X-Gal staining. To assess the utility of polyPOZ as a tool for confirming the cell identity of expressing and non-expressing cell types in the ‘speckled’ and Wnt-1myc expressing glands, we used immunohistochemistry combined with electron microscopy (Fig. 5v-vii). In these studies, X-Gal was detected due to the presence of its electron dense crystal precipitate. Smooth muscle actin was simultaneously detected using nanogold-coupled antisera. Studies of the ‘speckled glands’ clearly showed the presence of transduced luminal cells as identified by the presence of microvilli, while other regions showed the presence of transduced myoepithelial cells. In the Wnt-1myc hyperplasia, expression of X-Gal was detected in myoepithelial cells and within the poorly structured epithelial cells (Fig.5vii).

Fig. 5.

Histological analysis of transplanted glands. Identification of retrovirally transduced cells using light (i-iv) and electron microscopy (v-vii). Following whole-mount analysis, glands were sectioned and analysed for X-Gal staining and smooth muscle actin expression. (i) X-Gal expression in polyPOZ control transduced ‘speckled’ gland (compare with Fig. 4Bviii; bar, 35 μm); (ii) polyPOZ control transduced ‘solid’ gland (compare with Fig. 4Bvi; bar, 100 μm); (iii) polyPOZ-Wnt-1myc transduced gland (non-expressing region) stained for smooth muscle actin (bar, 100 μm); (iv) Wnt-1myc transduced gland stained for smooth muscle actin (compare with Fig. 4Bii; bar, 50 μm); (v) smooth muscle actin localisation in non-expressing region of Wnt-1myc infected gland; f, fibroblast; me, myoepithelial cell (bar, 5 μm); (vi) X-Gal localisation in polyPOZ-transduced ‘speckled gland’ (compare with Fig. 4Bviii; bar, 2 μm); (vii) smooth muscle actin and X-Gal localisation in Wnt-1myc transduced gland; arrowhead, X-Gal crystals; arrow, lumen as indicated by microvilli (compare with Fig. 4Bii; bar, 2 μm).

Fig. 5.

Histological analysis of transplanted glands. Identification of retrovirally transduced cells using light (i-iv) and electron microscopy (v-vii). Following whole-mount analysis, glands were sectioned and analysed for X-Gal staining and smooth muscle actin expression. (i) X-Gal expression in polyPOZ control transduced ‘speckled’ gland (compare with Fig. 4Bviii; bar, 35 μm); (ii) polyPOZ control transduced ‘solid’ gland (compare with Fig. 4Bvi; bar, 100 μm); (iii) polyPOZ-Wnt-1myc transduced gland (non-expressing region) stained for smooth muscle actin (bar, 100 μm); (iv) Wnt-1myc transduced gland stained for smooth muscle actin (compare with Fig. 4Bii; bar, 50 μm); (v) smooth muscle actin localisation in non-expressing region of Wnt-1myc infected gland; f, fibroblast; me, myoepithelial cell (bar, 5 μm); (vi) X-Gal localisation in polyPOZ-transduced ‘speckled gland’ (compare with Fig. 4Bviii; bar, 2 μm); (vii) smooth muscle actin and X-Gal localisation in Wnt-1myc transduced gland; arrowhead, X-Gal crystals; arrow, lumen as indicated by microvilli (compare with Fig. 4Bii; bar, 2 μm).

Murine 10T1/2 fibroblasts were transduced with polyPOZ (C), polyPOZ-Wnt-1myc (1), polyPOZ-Wnt-5bmyc (5b) and polyPOZ-Wnt-7bmyc (7b) retroviruses. The left column shows Giemsa staining and the right column shows X-Gal stained cells. Bars, 40 μm. (ii) PolyPOZ-Wnt-1myc and polyPOZ-Wnt-7bmyc alter 10T1/2 fibroblast proliferation. 10T1/2 fibroblasts were plated in 96-well dishes at a range of densities and transduced at an MOI of 5 with control and Wnt-expressing polyPOZ retroviruses. Following 8 days of culture, cell proliferation was measured using CellTiter 96 assay from Promega.

This report shows that the hyperplastic effects of Wnt-1 expression in the mammary gland are not shared by Wnt-7b, despite each having similar effects on the C57MG mammary epithelial cell line. We demonstrated expression in vivo of the polyPOZ-Wnt-7b retrovirus and showed that it was biologically active in murine 10T1/2 fibroblasts. Transduction with the same polyPOZ-Wnt-7bmyc retrovirus repressed tooth development in murine mandibular arch explants (Sarkar et al., 2000). Previous studies of retroviral-driven Wnt expression in vivo showed that Wnt-4 did induce a mammary hyperplasia, rather similar to the hyperplasia induced by Wnt-1, even though it did not transform C57MG mammary epithelial cells (Bradbury et al., 1995; Wong et al., 1994). In conjunction, these results show that Wnts can have different activities in vivo and in vitro: Wnt-4 is poorly or not transforming and induces hyperplasias in vivo, while Wnt-7b transforms but has no detectable activity in the mammary gland in vivo. While the C57MG transformation assay does distinguish between Wnt family members, the induction of a mammary hyperplasia in vivo may be more relevant in assessing the outcome of the Wnt overexpression that occurs in breast tumours (Dale et al., 1996; Huguet et al., 1994; Lejeune et al., 1995).

The present study also failed to find changes associated with the expression of Wnt-5bmyc, but this result was inconclusive since, in the absence of a biological assay for Wnt-5bmyc activity, we cannot prove that our construct is functionally active.

The oncogenic effects of Wnt-1 have been suggested to reflect the inappropriate activation of Wnt signal transduction pathways that regulate normal mammary gland development (Buhler et al., 1993; Gavin and McMahon, 1992; Weber-Hall et al., 1994). Unlike Wnt-1, the other Wnts used here, i.e. Wnt-4, Wnt-5b and Wnt-7b, are expressed during normal mammary gland development; however, only Wnt-4 was found to induce significant morphological changes when expressed in vivo (Bradbury et al., 1995). There could be several reasons for these differences. For example, the response of the gland to endogenous Wnt-7b may be saturated with respect to ligand. Wnt-7b was found to be expressed in primary mammary epithelial cells, but not in epithelial-free mammary fat pads (C. Niemeyer and T. C. Dale; unpublished observation), strongly suggesting that Wnt-7b is normally expressed in the epithelium. The lack of response to Wnt-7b expression may also reflect the competence of the epithelium. For example, Wnt-7b may normally function at earlier or later times of mammary gland development. We have not studied Wnt-7b expression in the foetal mammary gland, but studies of Wnt-7bmyc expressing glands during early pregnancy failed to reveal differences from control glands.

Irrespective of the normal role of Wnt-7b, the retroviral studies show that ectopic Wnt-7b expression has a different outcome from that of Wnt-1. Reasons for this difference may include the specificity of the mammary Frizzled receptors for Wnt-7b compared with Wnt-1, or the presence of soluble Wnt inhibitors with differential ligand specificity (Wang et al., 1997). At present, these possibilities cannot be adequately explored as the ligand-receptor specificity profiles have not been fully established and the distribution of Wnt receptors and inhibitors has not been characterised during mammary gland development.

Staining of tissues to monitor β-galactosidase expression is a commonly used method to mark cells expressing genes of interest (MacGregor et al., 1991). In this study, the technique was further developed to allow the sequential analysis of stained mammary tissues at the whole mount, light and electron microscopy scales. In conjunction with the polyPOZ retrovirus, this approach should prove generally useful in the analysis of gene function. In particular, electron microscopy enables the correlation of cell type and ultrastructural pathology with the level of expression of the gene of interest. As previously documented, Wnt-1 induced a mammary hyperplasia. The use of the polyPOZ retrovirus has extended this analysis to show that the effects of Wnt-1 localised closely to expressing regions of the duct and that the extent of abnormality correlated with the level of retroviral expression. polyPOZ-Wnt-1myc was detected in both myoepithelial and luminal epithelial cells and caused a range of pathologies. At the whole-mount level, Wnt-1 induced an increase in ductal branching and a pregnancy-like morphology. While the overall structure of the Wnt-1-expressing ducts was disrupted, closer analysis showed that the normal apposition of luminal and myoepithelial cells was often maintained; however, some regions expressing high levels of X-Gal contained abnormal clusters of cells with either myo or luminal epithelial character, suggesting that Wnt-1 can also disrupt the myo-luminal epithelial interaction.

Analysis of X-Gal staining patterns in a number of control polyPOZ-transduced glands identified two distinct patterns: the solid and the speckled gland (Fig. 4Bvi-viii). Within each pattern, there was little cell to cell variability in the level of retroviral expression. In principle, non-expressing cells may lack the retrovirus or may have inactivated retroviral expression. We cannot at present exclude the second of these possibilities; however, the data is consistent with recent studies of mammary stem cells (Kordon and Smith, 1998; Smith, 1996). These studies identifed three stem cell potentials: ductal only (D), lobular only (L) and ductal-lobular (D-L). Interestingly, all three clone types generated both myo and luminal epithelial cells; however, the size of the outgrowths from D or L progenitors was small by comparison with the D-L progenitors. On the basis of the size of the epithelial outgrowths, we suggest that the extensive areas of solid X-Gal expressing gland were generated by transduction of a D-L stem cell. By contrast, the more limited areas occupied by ‘speckled’ areas may represent transduction of a D progenitor in which more than one progenitor collaborates to make a section of duct. Alternatively, the speckled gland may originate from an L progenitor that becomes distributed at future sites of lobular development throughout the duct. Future analyses using β-galactosidase-expressing recombinant retroviruses should help distinguish between these possibilities.

The clinical importance of Wnt signalling in the breast is becoming clearer as Wnt overexpression and deregulated Wnt signal transduction are uncovered in mammary cancer (Dale et al., 1996; Huguet et al., 1994; Lejeune et al., 1995; Roose et al., 1999). Studies of model systems including Drosophila melanogaster and Xenopus have shown that normal development involves the coordinate functions for ligands of the Wnt, FGF, TGF, hedgehog and EGF peptide families. The biological effects of members of these families on mammary gland development (Daniel and Robinson, 1992; Jackson et al., 1997; Niemann et al., 1998; Yang et al., 1995) suggest that normal breast development and oncogenesis may involve similar coordinate or deregulated activation of these ligands and their signal transduction pathways. Expression of these genes in vivo, together with an analysis of mammary stem cell lineage using vectors such as polyPOZ virus, should aid the understanding of these processes.

Abram
,
C. L.
,
Page
,
M. J.
and
Edwards
,
P. A.
(
1997
).
A new retroviral vector, CA1, to identify and select for cells expressing an inserted gene in vitro and in vivo
.
Gene
196
,
187
189
.
Adam
,
M. A.
,
Ramesh
,
N.
,
Miller
,
A. D.
and
Osborne
,
W. R.
(
1991
).
Internal initiation of translation in retroviral vectors carrying picornavirus 5′ nontranslated regions
.
J. Virol
.
65
,
4985
4990
.
Banerjee
,
M. R.
,
Wood
,
B. G.
,
Lin
,
F. K.
and
Crump
,
L. R.
(
1976
).
Organ culture of whole mammary gland of the mouse
.
Tissue Culture Ass. Manual
2
,
457
462
.
Boutros
,
M.
,
Paricio
,
N.
,
Strutt
,
D. I.
and
Mlodzik
,
M.
(
1998
).
Dishevelled activates JNK and discriminates between JNK pathways in planar polarity and wingless signaling
.
Cell
94
,
109
118
.
Bradbury
,
J. M.
,
Sykes
,
H.
and
Edwards
,
P. A. W.
(
1991
).
Induction of Mouse Mammary Tumours in a Transplantation System by the Sequential Introduction of the Myc and Ras Oncogenes
.
Int. J. Cancer
48
,
908
914
.
Bradbury
,
J. M.
,
Niemeyer
,
C. C.
,
Dale
,
T. C.
and
Edwards
,
P. A. W.
(
1994
).
Alterations of the growth characteristics of the fibroblast cell line C3H 10T1/2 by members of the Wnt gene family
.
Oncogene
9
,
2597
2603
.
Bradbury
,
J. M.
,
Edwards
,
P. A. W.
,
Niemeyer
,
C. C.
and
Dale
,
T. C.
(
1995
).
Wnt-4 expression induces a pregnancy-like growth pattern in reconstituted mammary glands in virgin mice
.
Dev. Biol
.
170
,
553
563
.
Brown
,
A. M.
,
Wildin
,
R. S.
,
Prendergast
,
T. J.
and
Varmus
,
H. E.
(
1986
).
A retrovirus vector expressing the putative mammary oncogene int-1 causes partial transformation of a mammary epithelial cell line
.
Cell
46
,
1001
1009
.
Buhler
,
T. A.
,
Dale
,
T. C.
,
Kieback
,
C.
,
Humphreys
,
R. C.
and
Rosen
,
J. M.
(
1993
).
Localization and quantification of Wnt-2 gene expression in mouse mammary development
.
Dev. Biol
.
155
,
87
96
.
Cepko
,
C.
and
Pear
,
W.
(
1995
).
Preparation of a specific retrovirus producer cell line
.
In Current Protocols in Molecular Biology
, vol.
1
(ed.
F. M.
Ausubel
,
R.
Brent
,
R. E.
Kingston
,
D. D.
Moore
,
J. G.
Seidman
,
J. A.
Smith
and
K.
Struhl
), pp.
9
.10.11-19.14.16:
John Wiley and Sons Inc
.
Cook
,
D.
,
Fry
,
M. J.
,
Sumatipala
,
R.
,
Hughes
,
K.
,
Woodgett
,
J. R.
and
Dale
,
T. C.
(
1996
).
Wingless inactivates glycogen synthase kinase-3 via an intracellular signalling pathway which involves a protein kinase C
.
EMBO J
.
15
,
4526
4536
.
Dale
,
T. C.
,
Weber-Hall
,
S. J.
,
Smith
,
K.
,
Huguet
,
E. L.
,
Jayatalike
,
H.
,
Gusterson
,
B. A.
,
Shuttleworth
,
G.
,
O’Hare
,
M.
and
Harris
,
A. L.
(
1996
).
Compartment switching of WNT-2 expression in human breast tumours
.
Cancer Res
.
56
,
4320
4323
.
Dale
,
T. C.
(
1998
).
Signal transduction by the Wnt family of ligands
.
Biochem. J
.
329
,
209
223
.
Daniel
,
C. W.
and
Robinson
,
S. D.
(
1992
).
Regulation of mammary growth and function by TGF-beta
.
Mol. Reprod. Dev
.
32
,
145
151
.
Edwards
,
P. A.
,
Ward
,
J. L.
and
Bradbury
,
J. M.
(
1988
).
Alteration of morphogenesis by the v-myc oncogene in transplants of mammary gland
.
Oncogene
2
,
407
412
.
Edwards
,
P. A. W.
,
Hiby
,
S. E.
,
Papkoff
,
J.
and
Bradbury
,
J. M.
(
1992
).
Hyperplasia of mouse mammary epithelium induced by expression of the Wnt-1 (int-1) oncogene in reconstituted mammary gland
.
Oncogene
7
,
2041
2051
.
Finch
,
P. W.
,
He
,
X.
,
Kelley
,
M. J.
,
Uren
,
A.
,
Schaudies
,
R. P.
,
Popescu
,
N. C.
,
Rudikoff
,
S.
,
Aaronson
,
S. A.
,
Varmus
,
H. E.
and
Rubin
,
J. S.
(
1997
).
Purification and molecular cloning of a secreted, Frizzled-related antagonist of Wnt action
.
Proc. Natl. Acad. Sci. USA
94
,
6770
6775
.
Gavin
,
B. J.
and
McMahon
,
A. P.
(
1992
).
Differential regulation of the Wnt gene family during pregnancy and lactation suggests a role in postnatal development of the mammary gland
.
Mol. Cell. Biol
.
12
,
2418
2423
.
Ghattas
,
I. R.
,
Sanes
,
J. R.
and
Majors
,
J. E.
(
1991
).
The encephalomyocarditis virus internal ribosome entry site allows efficient coexpression of two genes from a recombinant provirus in cultured cells and in embryos
.
Mol. Cell. Biol
.
11
,
5848
5859
.
Gonzalez
,
F.
,
Swales
,
L.
,
Bejsovec
,
A.
,
Skaer
,
H.
and
Martinez
,
A. A.
(
1991
).
Secretion and movement of wingless protein in the epidermis of the Drosophila embryo
.
Mech. Dev
.
35
,
43
54
.
He
,
T. C.
,
Sparks
,
A. B.
,
Rago
,
C.
,
Hermeking
,
H.
,
Zawel
,
L.
,
da Costa
,
L. T.
,
Morin
,
P. J.
,
Vogelstein
,
B.
and
Kinzler
,
K. W.
(
1998
).
Identification of c-MYC as a target of the APC pathway
.
Science
281
,
1509
1512
.
Hsieh
,
J. C.
,
Kodjabachian
,
L.
,
Rebbert
,
M. L.
,
Rattner
,
A.
,
Smallwood
,
P. M.
,
Samos
,
C. H.
,
Nusse
,
R.
,
Dawid
,
I. B.
and
Nathans
,
J.
(
1999
).
A new secreted protein that binds to Wnt proteins and inhibits their activities
.
Nature
398
,
431
436
.
Huguet
,
E. L.
,
McMahon
,
J. A.
,
McMahon
,
A. P.
,
Bicknell
,
R.
and
Harris
,
A. L.
(
1994
).
Differential expression of human Wnt genes 2,3,4 and 7B in human breast cell lines and normal and disease states of human breast tissue
.
Cancer Res
.
54
,
2615
2621
.
Jackson
,
D.
,
Bresnick
,
J.
,
Rosewell
,
I.
,
Crafton
,
T.
,
Poulsom
,
R.
,
Stamp
,
G.
and
Dickson
,
C.
(
1997
).
Fibroblast growth factor receptor signalling has a role in lobuloalveolar development of the mammary gland
.
J. Cell Sci
.
110
,
1261
1268
.
Kordon
,
E. C.
and
Smith
,
G. H.
(
1998
).
An entire functional mammary gland may comprise the progeny from a single cell
.
Development
125
,
1921
1930
.
Lejeune
,
S.
,
Huguet
,
E. L.
,
Hamby
,
A.
,
Poulsom
,
R.
and
Harris
,
A. L.
(
1995
).
Wnt5a cloning, expression and upregulation in human primary breast cancers
.
Clin. Cancer Res
.
1
,
215
222
.
Li
,
L.
,
Yuan
,
H.
,
Xie
,
W.
,
Mao
,
J.
,
Caruso
,
A. M.
,
McMahon
,
A.
,
Sussman
,
D. J.
and
Wu
,
D.
(
1999
).
Dishevelled proteins lead to two signaling pathways. Regulation of LEF-1 and c-Jun N-terminal kinase in mammalian cells
.
J. Biol. Chem
.
274
,
129
134
.
Lin
,
T. P.
,
Guzman
,
R. C.
,
Osborn
,
R. C.
,
Thordarson
,
G.
and
Nandi
,
S.
(
1992
).
Role of endocrine, autocrine, and paracrine interactions in the development of mammary hyperplasia in Wnt-1 transgenic mice
.
Cancer Res
.
52
,
4413
4419
.
Liu
,
P.
,
Wakamiya
,
M.
,
Shea
,
M. J.
,
Albrecht
,
U.
,
Behringer
,
R. R.
and
Bradley
,
A.
(
1999a
).
Requirement for Wnt3 in vertebrate axis formation
.
Nat. Genet
.
22
,
361
365
.
Liu
,
X.
,
Liu
,
T.
,
Slusarski
,
D. C.
,
Yang-Snyder
,
J.
,
Malbon
,
C. C.
,
Moon
,
R. T.
and
Wang
,
H.
(
1999b
).
Activation of a frizzled-2/beta-adrenergic receptor chimera promotes Wnt signaling and differentiation of mouse F9 teratocarcinoma cells via Galphao and Galphat
.
Proc. Natl. Acad. Sci. USA
96
,
14383
14388
.
MacGregor
,
G. R.
,
Nolan
,
G. P.
,
Fiering
,
S.
,
Roederer
,
M.
and
Herzenberg
,
L. A.
(
1991
).
Use of E. coli lacZ (β-galactosidase) as a reporter gene
.
In Methods in Molecular Biology, vol. 7
(ed.
E. J.
Murray
), pp.
217
235
.
Clifton, NJ
:
Humana Press
.
Markowitz
,
D.
,
Goff
,
S.
and
Bank
,
A.
(
1988
).
A safe packaging line for gene transfer: Separating viral genes on two different plasmids
.
J. Virol
.
62
,
1120
1124
.
Monkley
,
S. J.
,
Delaney
,
S. J.
,
Pennisi
,
D. J.
,
Christiansen
,
J. H.
and
Wainwright
,
B. J.
(
1996
).
Targeted disruption of the Wnt2 gene results in placentation defects
.
Development
122
,
3343
3353
.
Moon
,
R. T.
,
Brown
,
J. D.
and
Torres
,
M.
(
1997
).
WNTs modulate cell fate and behavior during vertebrate development
.
Trends Genet
.
13
,
157
162
.
Morgenstern
,
J.
and
Land
,
H.
(
1991
).
Choice and manipulation of retroviral vectors
.
In Methods in Molecular Biology
, vol.
7
(ed.
E. J.
Murray
), pp.
181
206
.
Clifton, NJ
:
Humana Press
.
Moriguchi
,
T.
,
Kawachi
,
K.
,
Kamakura
,
S.
,
Masuyama
,
N.
,
Yamanaka
,
H.
,
Matsumoto
,
K.
,
Kikuchi
,
A.
and
Nishida
,
E.
(
1999
).
Distinct domains of mouse Dishevelled are responsible for the c-Jun N-terminal Kinase/Stresss-activated Protein Kinase activation and the axis formation in vertebrates
.
J. Biol. Chem
.
274
,
30957
30962
.
Niemann
,
C.
,
Brinkmann
,
V.
,
Spitzer
,
E.
,
Hartmann
,
G.
,
Sachs
,
M.
,
Naundorf
,
H.
and
Birchmeier
,
W.
(
1998
).
Reconstitution of mammary gland development in vitro: requirement of c-met and c-erbB2 signaling for branching and alveolar morphogenesis
.
J. Cell Biol
.
143
,
533
545
.
Nusse
,
R.
,
Brown
,
A.
,
Papkoff
,
J.
,
Scambler
,
P.
,
Shackleford
,
G.
,
McMahon
,
A.
,
Moon
,
R.
and
Varmus
,
H.
(
1991
).
A new nomenclature for int-1 and related genes: the Wnt gene family (letter)
.
Cell
64
,
231
.
Nusse
,
R.
and
Varmus
,
H. E.
(
1982
).
Many tumours induced by the mouse mammary tumour virus contain provirus integrated in the same region of the host genome
.
Cell
31
,
99
109
.
Nusse
,
R.
and
Varmus
,
H. E.
(
1992
).
Wnt Genes. Cell
69
,
1073
1087
.
Orsulic
,
S.
and
Peifer
,
M.
(
1996
).
Cell-cell signalling: Wingless lands at last
.
Curr. Biol
.
6
,
1363
1367
.
Papkoff
,
J.
and
Schryver
,
B.
(
1990
).
Secreted int-1 protein is associated with the cell surface
.
Mol. Cell. Biol
.
10
,
2723
2730
.
Piccolo
,
S.
,
Agius
,
E.
,
Leyns
,
L.
,
Bhattacharyya
,
S.
,
Grunz
,
H.
,
Bouwmeester
,
T.
and
De Robertis
,
E. M.
(
1999
).
The head inducer Cerberus is a multifunctional antagonist of Nodal, BMP and Wnt signals
.
Nature
397
,
707
710
.
Polakis
,
P.
(
1999
).
The oncogenic activation of beta-catenin
.
Curr. Opin. Genet. Dev
.
9
,
15
21
.
Ramakrishna
,
N. R.
and
Brown
,
A. M. C.
(
1993
).
Wingless, the Drosophila homolog of the proto-oncogene Wnt-1, can transform mouse mammary epithelial cells
.
Development Supplement
,
95
103
.
Reichsman
,
F.
,
Smith
,
L.
and
Cumberledge
,
S.
(
1996
).
Glycosaminoglycans can modulate extracellular localization of the wingless protein and promote signal transduction
.
J. Cell Biol
.
135
,
819
827
.
Robertson
,
D.
,
Monaghan
,
P.
,
Clarke
,
C.
and
Atherton
,
A. J.
(
1992
)
An appraisal of low temperature embedding by progressive lowering of temperature into Lowicryl HM20 for immunocytochemical studies
.
J. Microsc
.
168
,
85
100
.
Roose
,
J.
,
Huls
,
G.
,
van Beest
,
M.
,
Moerer
,
P.
,
van der Horn
,
K.
,
Goldschmeding
,
R.
,
Logtenberg
,
T.
and
Clevers
,
H.
(
1999
).
Synergy between tumor suppressor APC and the beta-catenin-tcf4 target tcf1
.
Science
285
,
1923
1926
.
Sapino
,
A.
,
Macri
,
L.
,
Gugliotta
,
P.
and
Bussolati
,
G.
(
1990
).
Immunocytochemical identification of proliferating cell types in mouse mammary gland
.
J. Histochem. Cytochem
.
38
,
1541
1547
.
Sarkar
,
L.
,
Naylor
,
S.
,
Smalley
,
M.
,
Dale
,
T. C.
and
Sharpe
,
P. T.
(
2000
).
Interactions between Wnt-7b and Shh act to maintain boundaries between oral and dental ectoderm
.
Proc. Natl. Acad. Sci. USA, in press
.
Schryver
,
B.
,
Hinck
,
L.
and
Papkoff
,
J.
(
1996
).
Properties of Wnt-1 protein that enable cell surface association
.
Oncogene
13
,
333
342
.
Sheldahl
,
L. C.
,
Park
,
M.
,
Malbon
,
C. C.
and
Moon
,
R. T.
(
1999
).
Protein kinase C is differentially stimulated by Wnt and Frizzled homologs in a G-protein-dependent manner
.
Curr. Biol
.
9
,
695
698
.
Shimizu
,
H.
,
Julium
,
M. A.
,
Giarre
,
M.
,
Zheng
,
Z.
,
Brown
,
A. M. C.
and
Kitajewski
,
J.
(
1997
).
Transformation by Wnt family proteins correlates with regulation of b-catenin
.
Cell Growth Diff
.
8
,
1349
1358
.
Shtutman
,
M.
,
Zhurinsky
,
J.
,
Simcha
,
I.
,
Albanese
,
C.
,
D’Amico
,
M.
,
Pestell
,
R.
and
Ben-Ze’ev
,
A.
(
1999
).
The cyclin D1 gene is a target of the beta-catenin/LEF-1 pathway
.
Proc. Natl. Acad. Sci. USA
96
,
5522
5527
.
Slusarski
,
D. C.
,
Corces
,
V. G.
and
Moon
,
R. T.
(
1997
).
Interaction of Wnt and a Frizzled homologue triggers G-protein-linked phosphatidylinositol signalling
.
Nature
390
,
410
413
.
Smalley
,
M. J.
,
Titley
,
J.
and
O’Hare
,
M. J.
(
1998
).
Clonal characterization of mouse mammary luminal epithelial and myopeithelial cells separated by fluorescence-activated cell sorting
.
In Vitro Cell Dev. Biol
.
34
,
711
721
.
Smalley
,
M. J.
and
Dale
,
T. C.
(
1999
).
Wnt Signalling in mammalian development and tumourigenesis
.
Cancer Metast. Rev
.
18
,
215
230
.
Smith
,
G. H.
(
1996
).
Experimental mammary epithelial morphogenesis in an in vivo model: evidence for distinct cellular progenitors of the ductal and lobular phenotype
.
Breast Cancer Res. Treat
.
39
,
21
31
.
Stark
,
K.
,
Vainio
,
S.
,
Vassileva
,
G.
and
McMahon
,
A. P.
(
1994
).
Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4
.
Nature
372
,
679
682
.
Takada
,
S.
,
Stark
,
K. L.
,
Shea
,
M. J.
,
Vassileva
,
G.
,
McMahon
,
J. A.
and
McMahon
,
A. P.
(
1994
).
Wnt-3a regulates somite and tailbud formation in the mouse embryo
.
Genes Dev
.
8
,
174
189
.
Thomas
,
K. R.
,
Musci
,
T. S.
,
Neumann
,
P. E.
and
Capecchi
,
M. R.
(
1991
).
Swaying is a mutant allele of the proto-oncogene Wnt-1
.
Cell
67
,
969
976
.
Tsukamoto
,
A. S.
,
Grosschedl
,
R.
,
Guzman
,
R. C.
,
Parslow
,
T.
and
Varmus
,
H. E.
(
1988
).
Expression of the int-1 gene in transgenic mice is associated with mammary gland hyperplasia and adenocarcinomas in male and female mice
.
Cell
55
,
619
625
.
Wang
,
S.
,
Krinks
,
M.
and
Moos
,
M.
, Jr
. (
1997
).
Frzb-1, an antagonist of Wnt-1 and Wnt-8, does not block signaling by Wnts -3A, -5A, or -11
.
Biochem. Biophys. Res. Commun
.
236
,
502
504
.
Weber-Hall
,
S. J.
,
Phippard
,
D.
,
Niemeyer
,
C.
and
Dale
,
T. C.
(
1994
).
Developmental and hormonal regulation of Wnt gene expression in the mouse mammary gland
.
Differentiation
57
,
205
214
.
Whiting
,
J.
,
Marshall
,
H.
,
Cook
,
M.
,
Krumlauf
,
R.
,
Rigby
,
P. W.
,
Stott
,
D.
and
Allemann
,
R. K.
(
1991
).
Multiple spatially specific enhancers are required to reconstruct the pattern of Hox-2.6 gene expression
.
Genes Dev
.
5
,
2048
2059
.
Wodarz
,
A.
and
Nusse
,
R.
(
1998
).
Mechanisms of Wnt signaling in development
.
Ann. Rev. Cell Dev. Biol
.
14
,
59
88
.
Wong
,
G. T.
,
Gavin
,
B. J.
and
McMahon
,
A. P.
(
1994
).
Differential transformation of mammary epithelial cells by Wnt genes
.
Mol. Cell. Biol
.
14
,
6278
6286
.
Yamaguchi
,
T. P.
,
Bradley
,
A.
,
McMahon
,
A. P.
and
Jones
,
S.
(
1999
).
A Wnt5a pathway underlies outgrowth of multiple structures in the vertebrate embryo
.
Development
126
,
1211
1223
.
Yang
,
Y.
,
Spitzer
,
E.
,
Meyer
,
D.
,
Sachs
,
M.
,
Niemann
,
C.
,
Hartmann
,
G.
,
Weidner
,
K. M.
,
Birchmeier
,
C.
and
Birchmeier
,
W.
(
1995
).
Sequential requirement of hepatocyte growth factor and neuregulin in the morphogenesis and differentiation of the mammary gland
.
J. Cell Biol
.
131
,
215
226
.
Zhang
,
Y.
,
Neo
,
S. Y.
,
Wang
,
X.
,
Han
,
J.
and
Lin
,
S. C.
(
1999
).
Axin forms a complex with MEKK1 and activates c-Jun NH(2)-terminal kinase/stress-activated protein kinase through domains distinct from Wnt signaling
.
J. Biol. Chem
.
274
,
35247
35254
.