Ectodysplasin/NF-kappa B Promotes Mammary Cell Fate via Wnt/beta-catenin Pathway

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-kappa B in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-kappa B is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants.Peer reviewe

List of selected upregulated genes from the microarray.

<p>List of selected upregulated genes from the microarray.</p

NF-κB is required for the formation of Eda induced supernumerary mammary placodes.

<p>(A-C) Expression of <i>Tbx3</i> (A), <i>Wnt10b</i> (B) and <i>PTHrP</i> (C) was detected in the mammary buds of the WT, <i>K14-Eda</i>, <i>IκBαΔN</i> and compound <i>IκBαΔN</i>; <i>K14-Eda</i> embryos at E13.5. Arrows highlight the supernumerary mammary placodes in <i>K14-Eda</i> embryos. Note that no localized expression was visible between the 3^rd and the 4^th mammary bud in the compound <i>IκBαΔN</i>; <i>K14-Eda</i> embryos. (D) Scanning electron microscopy images of the mammary forming region confirmed absence of accessory mammary primordia in compound mutants. (Scale bar: 500 μm.)</p

Wnt pathway genes are upregulated already at E12.5 in the region where supernumerary mammary placodes form.

<p>Expression of (A) <i>Wnt10b</i>, (B) <i>Wn10a</i>, (C) <i>Kremen2</i>, (D) <i>Lgr4</i>, (E) <i>β-cat</i>, (F) <i>Tbx3</i>, and (G) <i>Nrg3</i> in <i>Eda</i>-/-, wild-type and <i>K14-Eda</i> embryos. Note correlation of levels of <i>Wnt10b</i>, <i>Wnt10a</i>, <i>Kremen2</i>, and <i>Lgr4</i> with the Eda status (A-D).Further, <i>Wnt10b</i>, <i>Kremen2</i>, <i>Lgr4</i>, and <i>β-cat</i> show an early stripe-like expression pattern in the region where supernumerary mammary placodes emerge (A, C-E). (Scale bar: 500 μm.)</p

Recombinant Eda protein induces supernumerary mammary placodes and upregulates NF-κB reporter expression ex vivo.

<p>(A-D) Stereomicroscope images of E12.5 flank skins of WT_C57BL/6 (n = 11) (A), <i>K14-Eda</i> (n = 15) (B), WT_NMRI (n = 21) (C) and WT_NMRI supplemented with 250 ng/mL of Fc-Eda (n = 42) (D) cultured for two days ex vivo. A and C are control littermates of B and D, respectively. Ectopic buds were observed in 0/32 control, 15/15 <i>K14-Eda</i>, and 38/42 Fc-Eda treated explants. (E-F) NF-κB reporter expression was analysed in E12.5+1d and E12.5+2d explants cultured in the control medium (E) and medium supplemented with 250ng/mL of Fc-Eda (F), by X-gal staining. (Scale bar: 100 μm.)</p

Inhibition of Wnt activity suppresses supernumerary mammary placode formation in a dose-dependent manner in <i>K14-Eda</i> tissue explants.

<p>(A-E) Stereomicroscope images of E12.5 TOP-gal (A) (n = 17), WT (B, D) and <i>K14-Eda</i> (C, E) explants cultured for 2 days in the presence of 10 μM XAV939 or equal amount of solvent (0.1% DMSO) (B, C), and 40 μM XAV939 or an equal amount of solvent (0.2% DMSO) (A, D, E). Supernumerary placodes were never observed in WT specimen (0/18 explants). (F) Quantification of supernumerary placodes in <i>K14-Eda</i> explants: data are shown as mean ±SD. ***P < 0.001, **P < 0.01. (B, C) WT _{(n = 10)} and <i>K14-Eda</i> _{(n = 10)}; (D, E) WT _{(n = 8)} and <i>K14-Eda</i> _{(n = 8)}. In all experiments, one side of the embryo was used as the control and the other was treated with XAV939. (Scale bar: 100 μm.)</p

<i>Edar</i> expression and NF-κB reporter expression co-localize in the mammary epithelium.

<p>(A) <i>Edar</i> transcripts were detected by in situ hybridization with a ³⁵S-UTP-labeled probe in mammary buds at E12.5 and E13.5. (B) NF-κB reporter was initially expressed throughout the mammary bud in control embryos, but became localized to the basal layer of the epithelium around E13.5. (C) In <i>K14-Eda</i> embryos, reporter activity stayed high throughout the mammary bud at E12.5 and E13.5. mb4 = mammary bud number 4. (D) The supernumerary buds exhibited mosaic expression of the reporter which was less pronounced in the in the neck (left) than in mammary primordia forming between buds 3 and 4 (right). (Scale bar: 100 μm.)</p

Supernumerary mammary gland in the neck consists of a nipple and a small ductal tree in <i>K14-Eda</i> mice.

<p>(A) Macroscopic view of the nipple of mammary gland 1 and two supernumerary nipples in the neck of a pregnant <i>K14-Eda</i> female. (B) Expression of the nipple marker keratin2e was readily observed in the endogenous but not in the supernumerary nipple. (C-E) Carmine alum stained ductal tree of mammary gland 1 and a supernumerary gland of K14-<i>Eda</i> female mouse at age of 5 weeks (C), on pregnancy day 14 (D), and day of parturition (E). (Scale bar: 500 μm)</p

Overexpression of Eda induces formation of supernumerary mammary placodes in the neck and the flank.

<p>(A-F) NF-κB reporter expression was analyzed by X-gal whole mount staining in control and <i>K14-Eda</i> littermates at E11.0 (A), 11.25 (B), E11.5 (C), E12.0 (D), E12.5 (E), and E13.5 (F). In control, a low level NF-κB activity was observed anterior to the fore limb bud whereas the same region in <i>K14-Eda</i> gave rise to supernumerary X-gal positive foci (arrowheads). By E12.5 NF-κB became downregulated in the inter-placodal region in the control embryos whereas in <i>K14-Eda</i> embryos the appearance of supernumerary placodes between buds 3 and 4 started to become apparent at E12.5 and was more pronounced at E13.5 (arrows). (G-H) Whole mount in situ hybridization using digoxigenin-labelled probes specific to <i>Wnt10b</i> (G) and <i>Dkk4</i> (H) at E11.25. Expression was detected in the emerging mammary placodes, as well as anterior to the fore limb bud (arrowheads) in both genotypes. (Scale bar: 500 μm).</p