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

(A-F) NF-κB reporter expression was analyzed by X-gal whole mount staining in control and K14-Eda 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 K14-Eda 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 K14-Eda 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 Wnt10b (G) and Dkk4 (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).

(A-F) 采用X-gal整体染色法，分析E11.0（图A）、11.25（图B）、E11.5（图C）、E12.0（图D）、E12.5（图E）及E13.5（图F）的对照组与K14-Eda同窝仔鼠体内NF-κB报告基因（NF-κB reporter）的表达情况。对照组小鼠前肢芽前方可观测到低水平的NF-κB活性，而K14-Eda组该区域则出现了额外的X-gal阳性灶（箭头标注）。至E12.5时，对照组胚胎的基板间区域NF-κB表达已出现下调；而K14-Eda胚胎中，第3与第4芽体间额外乳腺基板的形成在E12.5时初现端倪，于E13.5时表现更为显著（箭头标注）。(G-H) 在E11.25时，使用针对Wnt10b（G）与Dkk4（H）的地高辛（digoxigenin）标记特异性探针开展整体原位杂交（whole mount in situ hybridization）实验。两种基因型小鼠的新生乳腺基板以及前肢芽前方（箭头标注）均检测到了对应基因的表达。（比例尺：500 μm）