Specific and similar depletion of small RNA reads (19–23 nt) in both Dicer and Dgcr8 cKO skin. Small RNA reads from 18 to 28 nucleotides were charted from all four small RNA cDNA libraries. Note the nearly identical distribution patterns of small RNA reads within either two duplicate WT libraries or two cKO libraries.

Small RNA cloning and deep sequencing reveals the dependence of microRNA biogenesis on Dicer and DGCR8. (A) Compilation of microRNA sequencing data in Dicer and Dgcr8 cKO samples compared with their WT littermate skins reveal similar dependence of microRNA biogenesis with the exception for miR-320 and miR-484, which require Dicer but not DGCR8. MiR-21 was depleted in both Dicer and Dgcr8 cKO, but only partially (≈70%, circle). MicroRNA depletion ratios were normalized against reads from rRNA, tRNA, and spiked-in calibration RNAs, none of which depends on Dicer or DGCR8. (B) qPCR microRNA expression validation of the cloning/sequencing results. Of 67 skin microRNAs subjected to quantification by qPCR, 65 were dependent similarly on Dicer and DGCR8. MiR-320 and miR-484 displayed dependency on Dicer but not DGCR8.

Strategy for generating the Dgcr8 floxed allele in mouse genomic DNA (gDNA) and verification that microRNAs were effectively depleted as a result of Dgcr8 skin-specific ablation in mice. (A) Exon 3 within the Dgcr8 coding region was chosen for targeting because its deletion was predicted to generate a frameshift mutation and early truncation of the DGCR8 protein. Arrowheads denote positioning of the Lox sequences, recognized by Cre recombinase. WW, a WW protein–protein interaction module; dsRBD, double-stranded RNA-binding domain. (B) Conditional (K14-Cre) ablation of both Dgcr8 and Dicer in skin resulted in the depletion of microRNAs compared with their WT counterparts. The expression levels of three representative microRNAs highly expressed in skin were chosen for analyses. Values for WT samples were designated as 1. SnoRNA25 served as the internal control.

Skin phenotypes of Dgcr8 and Dicer cKO mice are strikingly similar. (A) Dgcr8 and Dicer cKO mice can survive up to 5–6 days after birth. Newborn (P0.5) cKO and WT mice are similar in size and appearance. Thereafter, cKO mice fail to gain weight and exhibit taught, flaky skin, a sign of severe dehydration. (B) P6 Dgcr8 and Dicer cKO skins display evaginating hair germs (hg) that appear as balls of undifferentiated cells (arrows) that distort the epidermis (epi). (Scale bar, 10 μm.) (C) Immunofluorescence identifies germs molecularly by transcription factor Lef1 and by hemidesmosomal β4 integrin (nonspecific dermal staining is caused by the secondary antibody). (Scale bar, 20 μm.) (D) Active caspase-3 (Cas3) denotes significant enrichment of apoptotic cells within hair germs in the cKO skin. Dotted lines mark epidermal-dermal boundaries. der, dermis. (Scale bar, 20 μm.)

Ultrastructural defects of Dicer and Dgcr8 cKO skin. (A) Skins from neonatal WT control, Dicer cKO and Dgcr8 cKO mice were fixed and processed for electron microscopy as described. Shown are regions of the epidermis, where a whorl of evaginated hair germ (hg) cells are readily identified by the presence of dermal papilla (DP) cells at the center of the structure. These aberrations in follicle morphogenesis distorted the surrounding epidermis. (Scale bar, 10 μm.) (B) Cells with hair follicle matrix cell (Mx) morphology were present in the evaginating hair germs of Dgcr8 cKO epidermis. These cells frequently contained melanin granules (Mel). Melanocytes are normally never in mouse skin epidermis, and the appearance of melanin within the whorls was an additional hallmark of the evaginating hair germs. (Scale bar, 2 μm.) (C and E) Closed-up pictures of boxed areas in A. Note the presence of hemidesmosomes (Hd) and a loose basal lamina (bl) at the evaginating hair germ-DP border (C) as well as the epidermal-dermal boundary to what appeared to be an underlying DP (E) in the Dgcr8 cKO skin. (Scale bar, 500 nm.) (D) Enriched apoptotic cells (Ap) in hair germs of Dgcr8 cKO skin. (Scale bar, 2 μm.) Additional abbreviations: SC, stratum corneum; Gr, granular layers; Sp, spinous layers; BL, basal epidermal layer; Der, dermis. Dotted lines mark epidermal-dermal boundaries.