Fig. 4. Ectopic Eda-A1 protein rescued the early Tabby skin phenotype and caused enlargement of the placodal fields in a quantitative manner when analysed with placode marker genes Shh or ß-catenin. Eda-deficient Tabby skin lacks the first hair follicles as shown by the absence of placode marker Shh whole-mount in situ (A). Shh expression in placodes was partially rescued by culturing the Tabby skin explants with 0.05 µg/ml of Fc-Eda-A1 protein (B). Full rescue was obtained with 0.1 µg/ml (C) to 0.5 µg/ml (D) of Fc-Eda-A1. Higher concentration (2 µg/ml) caused enlargement and fusion of the follicles as shown by Shh expression (E). Similarly, ß-catenin expression analysis showed rescue with 0.5 µg/ml of Fc-Eda-A1 (F) and fused follicles with 2 µg/ml (G). Wild-type E13 skin sample cultured for 1 day showed ß-catenin localisation to the forming hair placodes (H), while 2 µg/ml concentration of Fc-Eda-A1 protein (I) caused expansion and fusion of the ß-catenin expression domains of the wild-type skin. Skin explants prepared at E12 and cultured for 1 day did not show Shh expression (J) and no indication of placode formation was detected upon addition of 2 µg/ml Fc-Eda-A1 to the culture medium (K).