Figure 3. Dicer Mutant Skin at P7 Is Characterized by Lack of Shh and Notch1 Expression, Proliferative Defects, and Evagination of Dermal Cells into the Epidermis.

(A–F) Paraffin sections of skin from P7 Dicer mutant (B, D, and F) and littermate control (A, C, and E) subjected to immunofluorescence (red) for K14 (A and B), K1 (C and D), and S100A3 (E and F). Basal and suprabasal epidermal cell layers are expanded in the Dicer mutant. Differentiating hair-shaft cells are present in Dicer mutant hair follicles, but at lower numbers than in the control.

(G–N) Paraffin sections of skin from P7 Dicer mutant (H, J, L, and N) and littermate control (G, I, K, and M) subjected to in situ hybridization with the digoxygenin-labeled probes indicated. Positive signals appear purple brown and are indicated by arrows. Pigmented cells of the hair shaft appear black. Hybridization for Shh, Gli1, and Crabp1 was absent in Dicer mutant hair follicles, and Notch1 expression was absent from the mutant epidermis and severely reduced in hair follicles.

(O–P′) Immunofluorescence detection of BrdU in Dicer mutant (P and P′) and littermate control (O and O′) skin at P7. Labeled cells appear red. Their numbers are decreased in mutant hair follicles but increased in the epidermis compared with the control (arrows).

(Q and R) P7 Dicer mutant (R) and control (Q) skin subjected to TUNEL staining (green) and immunofluorescence for K17 (red). Note expanded K17 staining in the mutant epidermis. The frequency of TUNEL-positive cells is increased in Dicer mutant epidermis and hair follicles (arrows in [R]).

(S–X′) Immunoflourescence detection of GATA3 (S and T) (red), SOX9 (U and V) (red), K14 (W and X) (red), and K17 (W′ and X′) (green) in Dicer mutant (T, V, X, and X′) and littermate control (S, U, W, and W′) skin at P7. Note elevated SOX9 expression in Dicer mutant epidermis ([V], arrow) and evaginations of dermal cells into the epidermis, indicated by arrows in panels (V), (X), and (X′).

(Y–Z′) Littermate control (Y) and Dicer mutant (Z and Z′) P6.5 skin paraffin sections stained for alkaline phosphatase (AP) (purple). Note AP staining in control and mutant dermal papillae (arrows) and additional staining enclosed by epidermal evaginations (arrows in [Z] and [Z′]). Pigmented hair-shaft cells in (Y) appear black. Nuclei in panels (A)–(F), (O)–(X′), and (Z′) were counterstained with DAPI (blue). Panels (A)–(L), (O), and (P) were photographed at 10×; panels (Q), (R), (Y), and (Z) at 20×; and panels (M), (N), (O′), (P′), (S)–(X′), and (Z′) at 40×. DP denotes dermal papilla.