Figure 1. Expression of Dicer and miRNAs in the Skin and Generation of an Epidermal-Specific Dicer Deletion.

(A) E14.5 mouse embryo subjected to whole-mount in situ hybridization with Dicer probe. Generalized expression is detected in the epidermis (blue staining) with elevated signal in developing hair and whisker follicles (examples indicated by arrows).

(B) Section in situ hybridization of P7 dorsal mouse skin with Dicer probe. Intense signal (purple-brown) is detected in the epidermis and hair-follicle outer root sheath (arrows).

(C) Semiquantitative RT-PCR analysis of expression of miRNAs mmu-mir-200b, mmu-mir-196a, mmu-mir-29b, and mmu-mir-24 in mouse skin with primers specific for the mature miRNAs. The samples used were as follows: P6, isolated P6 epidermis; P16 ana, P16 posterior dorsal epidermis containing anagen-stage hair follicles; P16 Cat, P16 anterior dorsal epidermis containing catagen-stage hair follicles; P20, anterior dorsal P20 epidermis containing telogen-stage hair follicles; P1C, full-thickness P1 control dorsal skin; and P1 Dkk1, full-thickness dorsal skin from a P1 mouse ectopically expressing Dkk1 in the epidermis and lacking hair follicles.

(D) Schematic depiction of PCR primers used for detection of wild-type, floxed, and Cre-excised Dicer alleles.

(E) Analysis of dermal and epidermal genomic DNA isolated from newborn K14-Cre; Dicer^flox/+, Dicer^flox/+, and K14-Cre; Dicer^flox/flox mice. The Dicer^flox/+ allele is efficiently recombined in the epidermis, but not the dermis of mice carrying K14-Cre.

(F) Western blots of isolated dorsal epidermis at P1 (left panel) and full-thickness dorsal skin at P63 (right panel) from littermate control (cont) or Dicer mutant (KO) mice incubated with anti-Dicer antibody (upper panels), anti-p27 (bottom left), or anti-Ezrin (bottom right). Dicer protein is absent from KO epidermis at P1 and greatly reduced in full-thickness skin at P63.

(G) Northern blot of RNA from control and Dicer mutant epidermis at P1 (left panel) and P6 (right panel) hybridized with probe for mmu-mir-200b. miRNA precursors are detected in all lanes; the mature processed miRNA is detected in the control lanes but is absent from Dicer mutant epidermis.

(H) Semiquantitative RT-PCR analysis of expression of mmu-mir-27b, mmu-mir-203, mmu-mir-24, and mmu-mir-200b in control littermate and Dicer mutant skin with primers specific for the mature miRNAs. P6 RNAs were isolated from epidermis and P1 and P63 RNAs from full-thickness dorsal skin.

(I and J) Phenotypes of Dicer mutant mice with control littermates at P7 (I) and P13 (J). Note the complete lack of external hair in the Dicer mutant depicted at P7 (I). The mutant depicted at P13 showed a mosaic phenotype with loss of external hair over the majority of the body, but sparing the head and right flank.

(K–M) Whole mounts of dorsal skin from control littermate (K) or Dicer mutant (L and M) at P6 viewed from the epidermal (K and L) or dermal (M) side. Whole-mounted skin was overstained with alkaline phosphatase to reveal skin structure. Note epidermal evaginations in (L) (arrows) and failure of mutant hair shafts to penetrate the epidermis. Mutant hair follicles are misangled (M).

(N–V′) Histological analysis of Dicer mutant (O, O′, Q, Q′, S, T, V, and V′) and littermate control (N, N′, P, P′, R, U, and U′) skin from mice at P1 (N–O′), P7 (P–T), and P49 (U–V′). Paraffin sections were stained with hematoxylin and eosin. Arrow in (S) indicates abnormal stunted hair follicle in Dicer mutant skin at P7. Arrows in (T) indicate abnormal evaginations of dermal cells into Dicer null epidermis at P7. Panels (N), (O), (P), (Q), (U), and (V) were photographed at 10× magnification; (P′) and (Q′) at 20×; and (N′), (O′), (R)–(T), (U′), and (V′) at 40×. Size bar in panel (V′) indicates the following: 200 μm for panels (N), (O), (P), (Q), (U), and (V); 100 μm for panels (P′) and (Q′); and 50 μm for panels (N′), (O′), (R)–(T), (U′), and (V′).