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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Genesis. Author manuscript; available in PMC Jul 18, 2012.
Published in final edited form as:
PMCID: PMC3399184
NIHMSID: NIHMS389638
Dynamic Expression of a LEF-EGFP WNT Reporter in Mouse Development and Cancer
Nicolas Currier,1 Kathleen Chea,1 Mirka Hlavacova,1 Daniel J. Sussman,2 David C. Seldin,1 and Isabel Dominguez1*
1Department of Medicine, Boston University School of Medicine, Boston, Massachusetts
2New Horizons Diagnostics Corp., Columbia, Maryland
*Correspondence to: Isabel Dominguez, Ph.D., 650 Albany Street X430, Boston, MA 02118, USA. isdoming/at/bu.edu
We have characterized a transgenic mouse line in which enhanced green fluorescent protein (EGFP) is expressed under the control of multimerized LEF-1 responsive elements. In embryos, EGFP was detected in known sites of Wnt activation, including the primitive streak, mesoderm, neural tube, somites, heart, limb buds, mammary placodes, and whisker follicles. In vitro cultured transgenic embryonic fibroblasts upregulated EGFP expression in response to activation of Wnt signaling by GSK3β inhibition. Mammary tumor cell lines derived from female LEF-EGFP transgenic mice treated with the carcinogen 7, 12-dimethylbenz[a]anthracene (DMBA) also express EGFP. Thus, this transgenic line is useful for ex vivo and in vitro studies of Wnt signaling in development and cancer.
Keywords: Wnt reporter, EGFP, embryo, β-catenin, transgenic mouse, breast cancer
The Wnt signaling pathway was originally identified in Drosophila melanogaster as an important signaling pathway in wing development (Sharma and Chopra, 1976; Siegfried et al., 1994). Since then, Wnt signaling has been found to play a key role in cell fate regulation, organogenesis, and other fundamental developmental processes in many animal species. Wnts are a family of secreted glycoprotein ligands rich in cysteines that can signal through several pathways (Barker, 2008). Activation of the canonical Wnt/β-catenin pathway results in the stabilization of β-catenin, a transcriptional coactivator (Gordon and Nusse, 2006; Huang and He, 2008). Stabilization of β-catenin is mediated by its release from a “destruction” complex composed of casein kinase 1 (CK1), glycogen synthase kinase 3β (GSK3β), axin, and adenomatous polyposis coli (APC), as well as further stabilization by CK2 phosphorylation (Dominguez et al., 2004; Song et al., 2000, 2003; Wu et al., 2009). Stabilized β-catenin translocates into the nucleus, where it associates with DNA-binding factors of the lymphoid enhancer-binding factor (LEF) and T-cell factor (TCF) family. β-catenin/TCF complexes promote transactivation of developmental genes such as siamois (Brannon et al., 1997; Carnac et al., 1996) and brachyury (Arnold et al., 2000; Yamaguchi et al., 1999).
Beginning with the first reports of mammalian Wnt-1 inducing tumors in mice (Nusse and Varmus, 1982; Rijsewijk et al., 1987), research has shown that dysregulated canonical Wnt signaling plays a major role in tumorigenesis (Chen et al., 2008; Matushansky et al., 2008). Recent data suggest that Wnt signaling could be mediating some of the effects of carcinogen exposure in breast cancer, as elevated levels of β-catenin and CK2α are found in mouse mammary tumors from DMBA fed rats and mice (Landesman-Bollag et al., 2001; Currier et al., 2005). In addition, DMBA-derived tumors display increased levels of cyclin D1 (Currier et al., 2005), a Wnt/β-catenin target gene that is required for intestinal tumor formation in ApcMin mice (Ilyas, 2005). However, studies in mice suggest that cyclin D1 may not be required for breast tumor formation induced by Wnt signaling (Yu et al., 2001).
The lack of tissue-specific target genes to analyze endogenous activation of canonical Wnt signaling has prompted the development of Wnt reporters in different species (recently reviewed in Barolo, 2006) to determine if canonical Wnt signaling is involved in a particular function in cells or in vivo. In mice, several transgenic Wnt reporters with different promoters and numbers of TCF/LEF binding sites have been created (Table 1), either driving the expression of LacZ, the gene encoding β-galactosidase (DasGupta and Fuchs, 1999: p 31; Lustig et al., 2002: p 86; Maretto et al., 2003: p 9; Mohamed et al., 2004:p 35; Nakaya et al., 2005: p 68; Moriyama et al., 2007: p 10), or driving the expression of EGFP (Jho et al., 2002; Moriyama et al., 2007). Here we describe a LEF-EGFP reporter transgenic mouse that utilizes seven multimerized LEF-1 binding sites to drive expression of EGFP. The EGFP reporter allows us to directly visualize activation of the transgene during early embryonic development by fluorescence microscopy. In addition, this model allows us to test activation of β-catenin/TCF signaling in tumors derived from DMBA-fed mice and in vitro in cell lines analyzed by cell sorting.
Table 1
Table 1
Mouse Transgenic WNT Reporters
Generation of LEF-EGFP Reporter Mice
To identify tissues and cells in development and cancer in which β-catenin/TCF signaling is activated, we generated transgenic mice expressing EGFP under control of seven copies of a LEF-1-binding site derived from the pLEF7-fos-luciferase reporter construct (Hsu et al., 1998; Roth et al., 2004) (Fig. 1a). This reporter construct is a faithful Wnt-responsive reporter in vitro (Hsu et al., 1998; Sussman, 2002). The transgene construct contained a minimal c-fos promoter. Two LEF-EGFP transgenic FVB/N lines were created. The lines exhibited similar developmental expression patterns; thus, only one line was expanded for further detailed study. This line was bred to homozygosity and embryos were evaluated for EGFP expression at several stages of development. The presence of the transgene in embryos and adult mice was confirmed by PCR analysis of yolk sac or tail DNA, respectively. Embryos that were positive for the LEF-EGFP transgene DNA were confirmed to express EGFP protein by immunoblot (Fig. 1b).
FIG. 1
FIG. 1
LEF-EGFP reporter transgene. (a) Representation of the transgene construct with seven murine LEF-1 response elements inserted into the pEGFP-1 expression vector. (b) E12.5 embryo genotyping PCR for the LEF-EGFP transgene (top panel) and immunoblot analysis (more ...)
EGFP Reporter Gene Expression in Embryonic Tissues
Wnts are essential for axis formation and organogenesis during embryonic development. To evaluate how faithfully the LEF-EGFP is expressed in tissues known to respond to Wnt/β-catenin signaling during mouse embryonic development, we analyzed the pattern of expression of EGFP protein by fluorescence microscopy in isolated embryos at different stages, and we compared EGFP expression with the pattern of activated β-catenin and with other Wnt reporters (Table 1). In parallel, embryos isolated from wild type (WT) FVB mice were used as controls for tissue autofluorescence.
We found that EGFP was expressed in the posterior region of LEF-EGFP transgenic embryos from E6 to E8 (Fig. 2 and Supporting Information Fig. S1; embryos were staged according to Downs and Davies, 1993). EGFP was detected in the extraembryonic visceral endoderm (Supporting Information Fig. S1) in prestreak embryos, where nonphosphorylated β-catenin has also been found between E5.5–E6 (Mohamed et al., 2004). EGFP expression detected in the primitive streak and the mesoderm at streak and neural plate stages (Fig. 2 and Supporting Information Fig. S1) is similar to other Wnt-reporters at this stage (BAT-gal; Maretto et al., 2003, Axin2-lacZ; ten Berge et al., 2008, TCF/Lef-LacZ; Mohamed et al., 2004), and more extended than β-galactosidase activity described in TOPGal embryos (Merrill et al., 2004). EGFP fluorescence in the posterior of the streak embryo coincides with the location where non-phosphorylated β-catenin, a marker of active Wnt-signaling, is first detected in the embryo proper (Mohamed et al., 2004). Mesodermal EGFP fluorescence in embryos at early and late head fold stages extended as anteriorly in expression as in BATlacZ (Nakaya et al., 2005) and TCF/Lef-LacZ embryos (Mohamed et al., 2004) and more than in TOPGal embryos (Merrill et al., 2004). No fluorescence was detected in control embryos at these stages (Fig. 2 and Supporting Information Fig. S1, and data not shown).
FIG. 2
FIG. 2
LEF-eGFP reporter transgene expression in presomitic embryos. Bright-field next to corresponding UV images. (a) Early streak (ES, E6.75–7.25) lateral view; (b) late streak (LS, E7.25–7.75), lateral view; and (ce) E8 embryo: (c) (more ...)
Expression of EGFP changed dynamically during the early stages of somite formation (E8.0). At three somites, the strongest EGFP fluorescence was observed in the somites and presomitic mesoderm, followed by the lateral mesoderm; none was detected in control embryos (Fig. 3d). As embryos developed from three to seven somites (Supporting Information Fig. S2), faint EGFP fluorescence in the midbrain region intensified (Supporting Information Fig. S2) and EGFP fluorescence underneath the surface ectoderm close to the neural folds (Supporting Information Fig. S2a,b) expanded ventrally reaching the developing first pharyngeal arch (Supporting Information Fig. S2b,d,f,h). This pattern of EGFP expression is similar to that of migratory cranial neural crest cells reaching the first pharyngeal arch (Yoshida et al., 2008) which begins around the four to five somite stage with neural crest cells migrating from the midbrain (Serbedzija et al., 1992). EGFP fluorescence in E8 embryos was similar to the expression pattern in BATlacZ embryos (Dunty et al., 2008), although the EGFP expression is stronger in the somites than in presomitic mesoderm. EGFP fluorescence is not detected in the node (BAT-gal; Maretto et al., 2003, BATlacZ; Dunty et al., 2008) and it is less extensive than other reporters; for example TCF/Lef-LacZ, which is positive in the anterior portion of the embryo earlier (Lin et al., 2007; Mohamed et al., 2004), and Ax2/d2EGFP, which is expressed in the dorsal neural tube (Jho et al., 2002). A dynamic increase in EGFP fluorescence was also detected in the cardiac region as the heart tube forms (Supporting Information Fig. S2). EGFP fluorescence was detected in the heart tube and in a region dorsal and medial to the heart tube (Supporting Information Fig. S2). By comparison to staining in TCF/Lef-LacZ embryos (Lin et al., 2007), this medial region may correspond to the splanchnic mesoderm, where a portion of the cardiac progenitors reside (Cai et al., 2003).
FIG. 3
FIG. 3
LEF-eGFP reporter gene expression in E8 embryos. Bright-field next to corresponding UV images. (a) 3-somite embryo, ventral view; (b,c) 5-somite embryo, ventral and lateral view; (d) control embryo, ventral view at 7-somites. A: anterior; P: posterior; (more ...)
At E9.0, the strongest EGFP fluorescescence was observed in the somites, septum transversum, and unsegmented paraxial mesoderm (Fig. 4a). EGFP fluorescence was detected in the anterior part of the embryo consistent with the midbrain, the hindbrain, mesenchymal cells in the anterior part of the face, and the first and second pharyngeal arches. At 18 somites, staining around the ventral side of the optic vesicle was observed (Fig. 4 and Supporting Information Fig. S3). No fluorescence was detected in control embryos (Fig. 4a insert). The pattern of EGFP fluorescence was similar to the pattern of expression of other Wnt reporters such as BATgal and TOPgal (Brugmann et al., 2007), although less extended than that of TCF/Lef-LacZ (Lin et al., 2007; Mohamed et al., 2004). EGFP is detected throughout the somites and not exclusively in the dorsal portion as in (Maretto et al., 2003). At E9.5, strong EGFP fluorescence was observed in the somites, forelimb bud and unsegmented presomitic mesoderm (see Fig. 4). No fluorescence was detected in control embryos (Fig. 4b, insert). Changes in EGFP fluorescence were seen as somite number increased. EGFP fluorescence was detected in the forebrain (particularly in the diencephalon), midbrain (more strongly in the ventral region), midbrain/hindbrain boundary, and in cells in the hindbrain region (Fig. 4 and Supporting Information Fig. S3). EGFP fluorescent cells were observed in the anterior part of the face, around the optic vesicle, in the dorsal part of the otic vesicle and in the first, second, and third pharyngeal arches (Supporting Information Fig. S3). Dynamic EGFP fluorescence was also detected in the cardiac region as the heart tube formed (Supporting Information Fig. S3). EGFP positive cells became visible in the primitive ventricle and outflow tract at 16 somites, in the presumptive atria at 18 somites, and in a region dorsal and medial to the heart tube consistent with the splanchnic mesoderm at 19 somites. At 21 somites, additional fluorescence appeared in the conus region and in a region consistent with the proepicardium and epicardial migratory cells.
FIG. 4
FIG. 4
LEF-EGFP reporter gene expression in E9.0 and E9.5 embryos. (a) 16-somite (E9.0) embryo in bright-field and UV, with (i) magnified head, (ii) anterior somites, and (iii) tail bud. (b) 22-somite (E9.5) embryo in bright-field and UV, with (i) magnified (more ...)
At E10.5 and 11.5, EGFP was detected in the dorsal diencephalon, mesencephalon, mesencephalon/metencephalon boundary, and anterior metencephalon (Fig. 5 and not shown). Cranial expression also included the dorsal part of the otic and optic vesicles, the mandibular and maxillary components of the first pharyngeal arch and the second pharyngeal arch. EGFP was visible in the somites as well as the apical ectodermal ridge (AER) of both forelimb and hindlimb buds at E10.5 and 11.5 (Fig. 5 and not shown). No fluorescence was observed in tissues of control embryos (E10.5 shown in Fig. 5). At E10.5, otic vesicle, mandibular, maxillary, and AER expression is similar to that found in other TCF/β-catenin reporters. Areas with divergent expression include the brain, where the EGFP expression pattern is less extended than that of some Wnt reporters (e.g., midbrain and hindbrain region in BAT-gal embryos; Brugmann et al., 2007; Maretto et al., 2003), and dorsal neural tube in Ax2/d2EGFP embryos (Jho et al., 2002); the nasal folds, where EGFP is not detected compared to other reporters (TCF/Lef-LacZ mice; Mohamed et al., 2004 and BAT-gal embryos; Maretto et al., 2003) and the tail bud, where EGFP is less expressed than in other reporters including the ins-TOPEGFP (Moriyama et al., 2007).
FIG. 5
FIG. 5
LEF-EGFP reporter gene expression in E10.5 embryos. Bright-field next to corresponding UV images. (a) Lateral views of 34-somite embryos. Bright-field next to corresponding UV image. Magnified images of (b) the head, (c) the tail bud and (d) the fore (more ...)
In E12.5 and E13.5 embryos, additional sites of EGFP expression were detected (Fig. 6 and Supporting Information Fig. S4), further demonstrating the dynamic expression of the transgene during development. EGFP was detected in the whisker follicles and in the mesencephalon, as observed in other reporters (DasGupta and Fuchs, 1999; Moriyama et al., 2007); in the anterior and posterior of the pinna of the ear and in mesenchymal cells surrounding the forelimb bud at E13.5, similar to other reporters (Boras-Granic et al., 2006; Chu et al., 2004; Maretto et al., 2003; Moriyama et al., 2007); and in the five pairs of mammary placodes, as a ring of cells and in surrounding cells, as found by (Boras-Granic et al., 2006; Chu et al., 2004; Maretto et al., 2003; Moriyama et al., 2007). EGFP was also detected along the back of the embryo in a pattern we have not seen described elsewhere. EGFP expression in the AER of the fore and hindlimb buds, and in the tail bud (Supporting Information Fig. S4) is similar to other reporters. In these two tissues, some autofluorescent cells were observed in control embryos in regions overlapping the anterior and posterior portion of the limb buds and the ventral side of the tail bud (Supporting Information Fig. S4). In the coelomic cavity, EGFP positive cells were detected in two localized areas in the anterior part of the ovaries and in the dorsal side of the heart (Supporting Information Fig. S4). Around this stage, other Wnt reporters show expression in the atrioventricular endocardial cushions and the out-flow tract (Gitler et al., 2003). A tubular-like structure in the mesonephros in both males and females appear to be autofluorescent (Supporting Information Fig. S4). Hepatic fluorescence was limited to an unidentified subpopulation of cells present throughout the liver (Supporting Information Fig. S4), which was not reported in other Wnt reporter models. However, adult hepatocytes around the central veins are positive with the insTOP-reporters (Moriyama et al., 2007). A limited cell population in the lungs is EGFP positive and this bronchial pattern is consistent with cells of the nervous system (Supporting Information Fig. S4); this expression pattern was different from other Wnt reporters (Maretto et al., 2003).
FIG. 6
FIG. 6
LEF-eGFP reporter gene expression in E13.5 embryos. Magnified images of (a) the snout and whisker primordia, (b) the head, (c) outer ear (d) the forelimb region: white arrowhead: mammary placodes, black arrowhead: mesechyma surrounding forelimb bud. ( (more ...)
The LEF-EGFP reporter is a faithful Wnt reporter as EGFP fluorescence coincides with regions in the embryo where nonphosphorylated β-catenin is detected, such as the primitive streak (Mohamed et al., 2004). In addition, the expression pattern of LEF-EGFP closely overlaps that reported for TCF-1, TCF-4, and LEF-1 transcripts (Korinek et al., 1998; Oosterwegel et al., 1991). During organogenesis, the LEF-EGFP transgene is expressed in tissues shown to express Wnts such as the neural tube and heart (Joyner, 1996; Oosterwegel et al., 1991; van Genderen et al., 1994); developing somites (Ikeya and Takada, 1998); the AER (Hill et al., 2006; Kengaku et al., 1998; Oosterwegel et al., 1991); neural crests cells (Bastidas et al., 2004; Wu et al., 2005; Yanfeng et al., 2003); otic vesicle (Lovicu and McAvoy, 2005; Riccomagno et al., 2005); mammary placodes; and whiskers (Boras-Granic et al., 2006; Chu et al., 2004; Korinek et al., 1998).
The pattern of LEF-EGFP expression was similar to expression of other Wnt reporters although some divergences can be observed at E13.5. Differences in the extent of the expression of Wnt-reporters have been previously described (Brugmann et al., 2007; Nakaya et al., 2005), perhaps due to different staining times (Lin et al., 2007; Mohamed et al., 2004) and imaging techniques. Our model provides a well characterized additional reporter system that corroborates other observations. This model provides some particular advantages in terms of direct visualization of Wnt activation, including the high dynamic range of fluorescent detection, allowing visualization of both high and low expressing cells in a single embryo preparation, and the ability to isolate positive cells by FACS.
FACS Analysis of E13.5 and E9.5 Embryos
Consistent with the highly restricted EGFP expression pattern observed at E13.5 by fluorescence microscopy, the proportion of cells expressing EGFP as determined by flow cytometry comprised <0.3% of cells in single cell suspensions prepared from whole embryos (see Fig. 7), highlighting the extremely cell-specific activation of Wnt signaling at this stage. In contrast, the proportion of cells expressing EGFP as determined by flow cytometry in E9.5 embryos was ~ 37% (Supporting Information Fig. S5). This percentage corresponds with the broader extension of EGFP detected in fluorescence microscopy (Figure 4). Fluorescent microscopy or flow cytometry analysis allows for ex vivo studies, however, disadvantages of fluorescence may include attenuation of signal in larger organs or thicker tissue, or quenching by lipids. Autofluorescence of tissues can also be a disadvantage, in embryos and in adult tissues, for example in the mammary gland, where both transgenic and nontransgenic control mice exhibited autofluorescence during pregnancy, presumably due to lipid content of the glands (data not shown). These disadvantages can be overcome in part by examination of tissue sections by IHC or the use of in situ hybridization techniques, and by careful examination of nontransgenic controls.
FIG. 7
FIG. 7
Flow cytometry analysis of cells from E13.5 LEF-EGFP embryo (left panel) and wild-type control (right panel). EGFP-positive cells appeared within the gate. In three transgenic embryos, 0.29% ± 0.04% of cells were EGFP positive, while <0.05% (more ...)
EGFP Expression is Upregulated in Response to GSK-3b Inhibition and Correlates With β-Catenin Expression in Cultured Mouse Embryonic Fibroblasts (MEFs)
To determine whether the LEF-EGFP reporter responds to signals that upregulate the Wnt pathway, we generated primary mouse embryonic fibroblast (MEF) cell lines for in vitro studies. One way to activate the Wnt pathway in cells is by inhibition of the kinase GSK-3β using pharmacological agents. We have previously used the drug SB-216763 and shown that it is an efficient activator of the Wnt pathway (Farago et al., 2005). While DMSO-treated LEF-EGFP transgenic MEFs exhibited very little endogenous fluorescence (Fig. 8a), more than 80% of MEFs treated with SB-216763 were fluorescent. Control MEFs exhibited no fluorescence after treatment with either SB-216763 or DMSO (not shown). In parallel with the increased fluorescence, β-catenin and EGFP proteins assessed by immunoblot were increased with SB-216763 treatment compared to DMSO (Fig. 8b,c) corroborating activation of the Wnt pathway. Thus, cultured LEF-EGFP embryonic fibroblasts may be useful for understanding activation of Wnt signaling in vitro.
FIG. 8
FIG. 8
SB-216763-induced EGFP expression in cultured transgenic MEFs. MEFs generated from E13.5 LEF-EGFP embryos were exposed to 10 µM SB-216763 or DMSO vehicle control. (a) EGFP fluorescence is observed in SB-216763-treated cells but not DMSO-treated (more ...)
LEF-EGFP is Expressed in DMBA-Induced Mammary Tumor Cell Lines
We have previously reported that carcinogen-induced mammary tumors frequently have high levels of β-catenin and cyclin D1, suggesting that canonical Wnt signaling is activated in these tumors (Currier et al., 2005). To corroborate this, we treated LEF-EGFP transgenic mice with DMBA as in (Currier et al., 2005). Here, we were able to analyze fluorescence in mammary tumor cell lines derived from the transgenic mice. Two adenosquamous tumors were isolated and showed increased β-catenin and CK2 proteins by immunoblot (Fig. 9a) as in previous experiments in FVB/N mice (Currier et al., 2005). Cell lines were established from these tumors, and designated EGFP-D1.1 and EGFP-D1.2. The fluorescence of the cells derived from these tumors was assayed by flow cytometry. A nontransgenic mammary tumor cell line generated after exposure to DMBA, designated 3983, was used to control for autofluorescence. Both of the transgenic lines exhibited significantly more fluorescence than the control cell lines, consistent with constitutive Wnt pathway activation (Fig. 9b,c). Thus, the LEF-EGFP mice provide further evidence that Wnt signaling is activated in carcinogen-induced mouse mammary tumors. Our experiments in epithelial cell lines show that β-catenin and cyclin D1 activation depends upon CK2 activity (Song et al., 2000, 2003; Wu et al., 2009). Thus, it is plausible that the elevated levels of β-catenin and cyclin D1 in carcinogen-induced tumors are also CK2-dependent. The EGFP-D1.1 and EGFP-D1.2 (Lou et al., 2008) tumor lines together with conditional ablated CK2 mice will be useful for further studies of Wnt regulation in mammary tumorigenesis.
FIG. 9
FIG. 9
Expression of EGFP in mammary tumor cell lines from LEF-EGFP transgenic mice. DMBA-induced mammary tumors in LEF-EGFP mice have elevated expression of canonical Wnt signaling components. (a) Whole cell extracts from DMBA-induced LEF-EGFP mammary tumors (more ...)
In summary, the LEF-EGPF Wnt reporter is a useful model for embryonic and tumorigenesis studies concerning Wnt signaling.
Breeding and Genotyping of LEF-EGFP Transgenic Mice
The pLEF-EGFP transgene construct was generated by inserting a 400 bp Eco RI/Bam HI fragment containing seven multimerized wild-type LEF-1 binding sites and a minimal c-fos promoter (derived from a Xba I/Kpn I fragment of pLEF-fos-luciferase, generously provided by Dr. Grosschedl (University of California, San Francisco; Hsu et al., 1998; Roth et al., 2004) into the Eco RI/Bam HI sites of pEGFP-1 (Clontech). The LEF-EGFP transgene was prepared for injection by digesting pLEF-EGFP with Afl I/Xho I, isolating the 1.45-kb fragment by electrophoresis in a 1% low melting point agarose gel, and purifying the fragment using GENECLEAN II (Bio101) according to the manufacturers protocol. Transgenic mice were generated in the core facility at the University of Maryland; two FVB lines were bred. Genotyping was confirmed by PCR analysis by amplifying 659-bp fragment of the transgene EGFP sequence (sense 5′-ATG GTG AGC AAG GGC GAG GAG -3′ and antisense 5′-GAC CAT GTG ATC GCG CTT CTC -3′) was performed in an iCycler (Bio-Rad, Hercules, CA) with 35 cycles × 95°C for 40 s, 55°C for 50 s, and 72°C for 1 min 20 s.
Embryo Collection and Fluorescence Microscopy
LEF-EGFP mice were housed as described (Currier et al., 2005). LEF-EGFP mice pairs were mated and females examined daily for vaginal plugs (presence of the plug was considered as embryonic day E0.5). Isolated embryos were placed in cold 1× PBS; presomitic embryos were staged according to (Downs and Davies, 1993) and somitic embryos according to the number of somites. The number of transgenic embryos examined included 3 at ~ 6.5, 19 at ~ E7.5, 11 at ~ E8.5, 24 at ~ E9.5, 36 at ~ E10.5, 8 at ~ E11.5, 7 at ~ E12.5, and 22 at ~ E13.5. For controls, we examined 6 at ~ E7.5, 7 at ~ E8.5, 14 at ~ E9.5, 24 at E10.5, 12 at ~ E11.5, 9 at ~ E12.5, 12 at ~ E13.5. Representative embryos at different stages of development were photographed using a fluorescent stereomicroscope (Olympus SZX16) fitted with a digital camera (QImaging RETRIGA-2000RV, Fast1394) and QCapture software (QCaptureSuite 2.95.0 2007). Exposure times for whole embryo photographs were 3 s at E7.5, 5 s at E8.5, 4 s at E9.5, 1.5 s at E10.5, and 13.6 s at E13.5. Photographs were all pseudo-colored green. Only magnified photos were adjusted by increasing brightness and contrast to enhance EGFP visibility. Some embryos were snap-frozen on dry-ice and stored at −80°C for further analysis.
Embryonic Fibroblast Cell Culture
E13.5 embryos were placed in sterile cold 1× PBS, head and visceral organs removed, and embryos mechanically minced with a sterile razor blade, digested by incubating in 0.25% trypsin (Mediatech, Herndon, VA) for 5 min at 37°C, then homogenized to create a single cell suspension. The suspension was taken up in DMEM (Mediatech), 12.5% fetal bovine serum (SeraCare Life Sciences, Milford, MA), 50 U ml−1 penicillin 50 mg ml−1 streptomycin (Mediatech), and 4 mM l-glutamine (Mediatech), and filtered through a 70-µm filter (B.D, Lexington, KY) and grown in the same media at 37°C.
DMBA Treatment and Tumor Cell Culture
Ten virgin female LEF-EGFP mice were each given 6 weekly 1.0-mg dose of DMBA in 0.2 ml of sesame oil by oral gavage, beginning at 5 weeks of age (Currier et al., 2005; Trombino et al., 2000). Mice were then followed until either tumors developed or the mice died. Mice bearing tumors >0.5 cm in diameter were euthanized; mammary tumors and grossly normal mammary glands were excised, and portions of the tissues were prepared for histology. To establish cell lines derived from the tumors, a portion of the tumors was isolated, fragmented with a sterile razor blade under sterile conditions and digested by incubating in 0.25% trypsin/EDTA for 5 min at 37°C. The digested tumors were then added to DMEM, 10% fetal bovine serum, 50 U ml−1 penicillin 50 mg ml−1 streptomycin, and 4 mM l-glutamine, and filtered through a 70-µm cell filter, and cells were grown in the same media at 37°C. Two tumor cell lines, “EGFP-D1.1” and “EGFP-D1.2” (derived from Tumor 1 and 2, respectively) were generated. The remaining tissue was frozen on dry ice and stored at −80°C for molecular and biochemical studies.
Cell Culture and Treatment of Cell Lines
Mouse embryonic fibroblast (MEF) cell lines were grown in DMEM, 12.5% fetal bovine serum, 50 U ml−1 penicillin, 50 mg ml−1 streptomycin, and 4 mM l-glutamine. EGFP-D1.1 cell line was grown in DMEM/F12 (Gibco Invitrogen, Carlsbad, CA), 5% horse serum (Gibco Invitrogen), 50 U ml−1 penicillin 50 mg ml−1 streptomycin, 5 mg insulin (Sigma-Aldrich, St. Louis, MO), 0.25 mg hydrocortisone (Sigma-Aldrich), and 0.01 mg EGF. EGFP-D1.2 cell line was grown in DMEM, 10% fetal bovine serum, 50 U ml−1 penicillin 50 mg ml−1 streptomycin, and 4 mM l-glutamine. To control for autofluorescence and to reduce the number of mice used, a preexisting cell line was used, rel-3983D (Shin et al., 2006).
The GSK3 inhibitor, SB-216763 (Sigma-Aldrich) was dissolved in DMSO (Sigma-Aldrich) to a concentration of 10 mM, and stored at −20°C. MEFs, EGFP-D1.1, EGFP-D1.2, and rel-3983D cells were cultured on six-well plates for 24 h before SB-216763 treatment, and then cell media was supplemented with 10 mM SB-216763 or DMSO at 1:1,000 dilution to achieve a final drug concentration of 10 µM. Cells were treated for the indicated time, and photographed on a Nikon fluorescence microscope (Melville, NY) fitted with a digital camera (Hamammatsu Diagnostic Instruments, Sterling Heights, MI). The software used was OpenLab (Improvision, Waltham, MA).
Flow Cytometry
Cultured embryonic fibroblasts or mammary tumor cell lines were detached using either 0.25% trypsin EDTA solution (Mediatech) or Hyqtase (Hyclone, Logan, UT), respectively for 5 min at 37 or 20°C, respectively. Cells were then placed in complete media and centrifuged at 1,200 rpm for 5 min at 4°C. Supernatant was removed and cells were reconstituted in 1× PBS, 2% fetal bovine serum, and incubated for 30 min in 20 µg ml−1 propidium iodide (PI, Sigma-Aldrich). Cells from digested embryos were washed in 1× PBS, incubated for 30 min in 20 µg ml−1 propidium iodide in PBS (PI, Sigma-Aldrich), diluted with twice the volume of PBS and filtered through a 70-µm filter. Cells were analyzed on a Becton/Dickinson FACscan (BD Biosciences, Franklin Lakes, NJ). Live cells were identified by exclusion of propidium iodide and their EGFP signal intensity was examined. The software used was CellQuest Pro (BD Biosciences).
Immunoblot Analysis
Protein extracts prepared by homogenizing frozen embryos, cells, or tumors in lysis buffer containing a cocktail of protease inhibitors, gel electrophoresis, transfer, immunobloting, band visualization, and quantitative analysis performed as previously described (Currier et al., 2005). Primary antibodies were: anti-β-catenin (B.D.), anti-β-actin (Sigma-Aldrich), anti-GFP (Santa Cruz), and CK2α (B.D.).
Statistics
P-values were obtained with the Student’s t-test using the statistical analysis package in Excel.
ACKNOWLEDGMENTS
The authors thank Patrick Hogan for mouse colony management and DMBA treatment of mice. They thank Eekhoon Jho for comments to the manuscript and the sharing of data. They thank Sophie Astrof, Rieko Ajima, Esther Landesman-Bollag, and Irene Roman for comments on the manuscript; Wellington Cardoso, Ken Albretch, Dorothy Pazin, Marganit Farago, Beth Hovey, Hao Wu, and Jennifer Ward for helpful discussions. They thank Karen Symes for the use of her fluorescent stereomicroscope in the early phases of this project, and Marina Malikova and Erin Smith for help with its use; Darrell Kotton, Letty Kwok, Matthew Rarick, Annabel Belkina, Irene Roman, and Gerald Denis for assistance with FACS analysis; Mitchell White for assistance with fluorescence microscopy and Taimur H. Khan for assistance with embryo dissection and photography. This project could not have been carried out without the availability of the core facilities at Boston University Medical Campus including the Lab Animal Sciences Core, the Flow Cytometry Core, and the Imaging Core established by the Department of Medicine. They thank the Technical Director of the Imaging Core, Michael Kirber, for his technical support and enthusiasm.
Contract grant sponsor: AHA; Contract grant number: SDG 0735521T; Contract grant sponsor: NCI; Contract grant number: R01 CA71796; Contract grant sponsor: NIEHS; Contract grant number: P01 ES11624; Contract grant sponsor: DOD; Contract grant number: BCRP DAMD17-03-1-0560; Contract grant sponsor: BUSM, Department of Medicine; Contract grant number: Pilot grant; Contract grant sponsor: Karin Grunebaum Cancer Research Foundation; Contract grant number: Junior Faculty Award.
Footnotes
Additional Supporting Information may be found in the online version of this article.
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