Fig. 5.

Opening of the canal involves a precocious programme of differentiation. (A-L) Frontal sections through human fetal samples. (A-C,E-K) Developing ear canal. (D,L) Adjacent cranial skin. (A) At late 8 weeks, the closed canal comprises basal K14 cells (green) and more differentiated K10 cells (red) at the centre. DAPI nuclear stain (blue). (B) By 12 weeks the whole canal is lined with K14- (green) and K10-positive (red) cells. (C) The central cells of the canal expressed the granular layer marker involucrin (green), adjacent to the keratin 10 layer (red). (D) The skin at the same stage had not yet formed an involucrin-positive layer (green), above the K10-positive cells (red). (E-H) Trichrome-stained sections. (E) The ear canal is starting to open by 14 weeks. (F) A clear gap, filled with sloughed off cells, is evident at 16 weeks. (G) High-power magnification view of boxed are in G, highlighting distinct morphology of the cells at the centre of the canal (asterisk). (H) High-power magnification view of boxed are in F, highlighting distinct morphology of the cells lining the open canal (asterisk), with sloughed off cells in the centre. (I) At 14 weeks, the cells at the centre of the primary canal expressed locricrin (green), indicating terminal differentiation of the canal epithelium. DAPI nuclear stain (blue). Asterisk labels central cells. (J) As the canal opened, its sides were labelled with locricrin (asterisks), which also labelled the sloughed off cornified layer. (K) At 14 weeks the locricrin-expressing layer was associated with high levels of TUNEL staining (brown), indicating programmed cell death (Eosin counterstain). (L) Loricrin is not expressed in the skin by 16 weeks but some expression is associated with forming hair follicles. DAPI nuclear stain (blue). Scale bars: 100 µm in A,C,K; 150 µm in B; 500 µm in E,F; 50 µm in D,G-J,L.