FIGURE SUMMARY
Title

Wnt4 is not sufficient to induce lobuloalveolar mammary development

Authors
Kim, Y.C., Clark, R.J., Pelegri, F., and Alexander, C.M.
Source
Full text @ BMC Dev. Biol.

(A) Diagram of the tetracyline-inducible Wnt4 transgene cassette. (more details are given in the results section) (B) Expression of Wnt4 and the luciferase reporter is regulated by doxycycline. 293T cells stably expressing rtTA were transfected with either TMILA-Wnt4 vector or parental TMILA vector. Transfected cells were incubated for 24 hrs with or without doxycycline (1 μg/ml), and cell lysates were prepared to measure luciferase activity. Expression of Wnt4 protein was assessed through Western blot analysis using a mouse Wnt4-specific antibody. (C) Southern blot analysis of tet-Wnt4 transgenic mice. Genomic DNA from a non-transgenic mouse was used as a negative control and spiked with TMILA-Wnt4 vector fragment to determine the approximate copy number of the integrated transgene (probes and digestion as described in Methods).

Characterization of doxycycline-inducible Wnt4 transgenic mice. (A) Luciferase reporter gene expression in tet-Wnt4/MMTV-rtTA transgenic mammary glands. To express Wnt4 specifically in mammary glands, tet-Wnt4A and B mice were mated with transgenic mice that express rtTA under the control of MMTV-LTR promoter (MMTV-rtTA). The subsequent virgin female littermates (3 months old) were administered doxycycline containing water (2 mg/ml) for 1 month. Mammary gland lysates were prepared, and the luciferase activity of 10 μg of total protein lysate was measured. N.T, non-transgenic; rtTA, MMTV-rtTA; W4A, tet-Wnt4A strain; W4B, independent strain tet-Wnt4B; bitransgenics are shown as W4A or B/rtTA. (B) Induction of Wnt4 mRNA expression in mammary glands in doxycycline-administered mice. Wnt4 mRNA level was quantified by real time quantitative PCR (see methods) using total RNAs from mammary glands of mice administered doxycycline for 1 month. The fold induction is indicated with respect to control glands (from 7 week old mice). For mRNA preparations from non, single and bi-transgenic glands, plus designations indicate the presence of the transgene, and the order is shown as follows, Wnt4A/rtTA. Data marked with an asterix were samples from mice in estrus. (C) Induction of Wnt4 protein expression in doxycycline-treated bitransgenic mammary epithelial cells. Mammary epithelial cells were prepared from non-transgenic (-/-) and bitransgenic (+/+) glands and cultured for 2 days. Cells were treated with doxycycline (1 μg/ml) for another 24 hrs and lysates were prepared for Western blot analysis. Lysates from expression vector-transfected 293T cells were used as positive controls.

Ectopic Wnt4 expression is not sufficient to induce hyper side-branching. To assess side-branching development, inguinal #4 mammary glands were dissected out from the above doxycyline-treated female mice (treated with doxycycline for 1 month at 3 months of age) and stained with Carmine solution. Scale bar = 1 mm.

Ectopic mouse Wnt4 expression induces a non-canonical Wnt phenotype during zebrafish development. (A) Gain of function phenotypes induced by mouse Wnt4 in zebrafish embryo. Various doses of mouse Wnt4 or Wnt1 mRNA was injected to fertilized zebrafish eggs, and the phenotype was assessed 24 hrs after injection. (β-galactosidase (lacZ) mRNA was used as a negative control.) The phenotypes were classified into 3 groups (severe, strong and mild) based on the complexity of phenotype. (B) Wnt4 induces gain of function phenotypes in a dose dependent manner. On the y-axis, % total embryos are shown with each (color-coded) phenotype (severe to mild, shown in A), after injection with the indicated dose of control, Wnt1 or Wnt4 constructs.

Acknowledgments
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