Skin defects in <i>Hdh</i>^neoQ50/<i>Hdh</i>^ex4/5 hypomorphic embryos.

Representative images of wild-type (WT) and HdhneoQ50/Hdhex4/5 animals, at E18.5. A) At E18.5, following toluidine-blue dye exclusion assay, WT animal shows a functional epidermal barrier but HdhneoQ50/Hdhex4/5 animal has a compromised epidermal barrier on the ventral surface. B) Immunoblot analyses of WT, Hdhex4/5/+, HdhneoQ50/+ and HdhneoQ50/Hdhex4/5 skin protein extracts (E18.5). Bands of Pro-filaggrin (pro-Flg, 2X-Flg, 3X-Flg) at 50–70 KDa and higher molecular weight bands and processed filaggrin (Flg) at 27 KDa were detected in all samples but were decreased in HdhneoQ50/Hdhex4/5 extracts. The bands of Loricrin (Lor) and alpha-tubulin (Tuba1) were similar in all samples. C) Bar graphs summarizing skin-extract protein immunoblot band signal-intensity (relative to WT), expressed as standard deviation of 3 biologic replicates using 2 mice each, as quantified by Image J software. Unpaired t-test. P value D) Representative images of cryostat-sectioned skin tissue, with DAPI stained nuclei to show reduced staining of the PCNA proliferation marker in HdhneoQ50/Hdhex4/5 skin. Green = PCNA staining; Blue = DAPI-stained nuclei. Dashed lines depict the cornified basal epidermal layer. All images were taken using a fluorescent 20X objective. Scale bars: 50μm E) Representative images of cryostat-sectioned skin tissue stained with TUNEL assay to quantify apoptotic cell death (arrows) in WT and HdhneoQ50/Hdhex4/5 embryos. Dashed lines depict the cornified basal epidermal layer. All images were taken using a phase-contrast 20X objective. Scale bars: 50μm.