Fig. 4. Inv-Cldn6 transgenic mice have a defective EPB. (A) ß-gal assays were performed to assess the integrity of the EPB of transgenic animals, as compared to the wild type, at different stages of development. Under normal conditions, an intact EPB exists at E17.5 days thereby preventing the penetration of substances such as X-gal. When X-gal enters the epidermis a striking blue staining results. At E16.5 neither the wild-type nor the transgenic embryos possessed an intact EPB, yet at E18.5 there was no penetration of X-gal through the EPB of the wild-type embryos while transgenic embryos remained blue. Even after birth, transgenic neonates did not possess an EPB. (B) In addition to the ß-gal assays, DPM measurements were performed to assess the trans-epidermal water loss of transgenic and wild-type neonates. In representative experiments, there was greater than a 3-fold increase in the dehydration of transgenic neonates as compared to the wild type further supporting the notion that the defective EPB causes the massive dehydration and death of the transgenic animals. (C-F) Isolated CE preparations of newborn transgenic and wild-type epidermis showed that the rigid, polygonal structure of the wild-type CEs (D and F) disappeared in the transgenic samples. Transgenic CEs were mostly rounded in shape (E) and overall less abundant (C) leading to the fragile, less compact nature of the stratum corneum that is observed.