Dynamic expression of a LEF-EGFP Wnt reporter in mouse development and cancer

Abstract

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 GSK3beta 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.

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 with a GFP antibody (middle panel) and β-actin, used as a loading control, (lower panel).

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 (c–e) E8 embryo: (c) anterior, (d) posterior and (e) lateral views. (f) Control wild-type embryos at early head fold (EHF) stage. A: anterior; P: posterior; al: allantois; fg: foregut pocket; nf: neural fold.

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; al: allantois; fg: foregut; ht: heart tube; lm: lateral mesoderm; mg: midgut; n: node; nf: neural folds; ps; primitive streak; psm: presomitic mesoderm; s: somite.

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 head, (ii) anterior somites, and (iii) tail bud. di: diencephalon; fb: forebrain; fl: forelimb bud; hb: hindbrain; ht: heart tube; mb: midbrain; otp: otic pit; otv: otic vesicle; ov: optic vesicle; p1: first pharyngeal arch; p2: second pharyngeal arch; p3: third pharyngeal arch; ps; primitive streak; psm: presomitic mesoderm; s: somite; st: septum transversum; tel: telencephalon.

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 and hindlimb. (e) Control 34-somite embryos. Bright-field next to corresponding UV image; di: diencephalon; fl: forelimb bud; hb: hindbrain; hl; hindlimb bud; mes: mesencehalon; otv: otic vesicle; ov: optic vesicle; p1: first pharyngeal arch; p2: second pharyngeal arch; s: somite; tel: telencephalon.

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. (e) Magnified image of the mesenchyme surrounding forelimb bud. (f) Magnified image of the mammary placodes. (g) Punctate staining along the back of the embryo. (h) Control E13.5 embryos, lateral view. Bright-field next to corresponding UV image. mes: mesencehalon; tel: telencephalon; wf: whisker follicles.

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% of cells from two littermate wild-type controls were detected in the gated region.

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 controls (left); corresponding brightfield images (right). (b) EGFP and β-catenin proteins are upregulated in SB-216763-treated MEFs by immunoblot; β-actin loading control is shown. (c) Relative amounts of induced EGFP (left) and β-catenin protein (right). Gray bars, DMSO-treated cells, Black bars, SB-216763-treated cells.

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 (Tumors 1 and 2) and normal LEF-EGFP mammary gland (Normal) were subjected to immunoblotting for CK2α, β-catenin, and β-actin as loading control. (b) Flow cytometry analysis of cell lines EGFP-D1.1 (gray line) and EGFP-D1.2 (black line) established from tumors induced in LEF-EGFP transgenic mice by treatment with DMBA, compared to a non-EGFP-transgenic rel-3983D mouse mammary tumor cell line (filled gray). (c) Average EGFP fluorescence of three replicates of non-EGFP-transgenic rel-3983D control cell line and EGP-D1.1 and 1.2. P values shown in histogram.