A skin microRNA promotes differentiation by repressing 'stemness' - PubMed (original) (raw)

A skin microRNA promotes differentiation by repressing 'stemness'

Rui Yi et al. Nature. 2008.

Abstract

In stratified epithelial tissues, homeostasis relies on the self-renewing capacity of stem cells located within the innermost basal layer. As basal cells become suprabasal, they lose proliferative potential and embark on a terminal differentiation programme. Here, we show that microRNA-203 is induced in the skin concomitantly with stratification and differentiation. By altering miR-203's spatiotemporal expression in vivo, we show that miR-203 promotes epidermal differentiation by restricting proliferative potential and inducing cell-cycle exit. We identify p63 as one of the conserved targets of miR-203 across vertebrates. Notably, p63 is an essential regulator of stem-cell maintenance in stratified epithelial tissues. We show that miR-203 directly represses the expression of p63: it fails to switch off suprabasally when either Dicer1 or miR-203 is absent and it becomes repressed basally when miR-203 is prematurely expressed. Our findings suggest that miR-203 defines a molecular boundary between proliferative basal progenitors and terminally differentiating suprabasal cells, ensuring proper identity of neighbouring layers.

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Figures

Figure 1

Figure 1. Spatiotemporal expression of miR-203 during skin development

a, Temporal induction of miR-203 as measured by cloning frequency. b, d, e, In situ hybridization reveals restriction of miR-203 to suprabasal, differentiating layers of skin. sb, suprabasal; bl, basal layer; epi, epidermis; der, dermis; sg, sebaceous gland; irs, inner root sheath; hf, hair follicle. Asterisks in e represent brown melanin pigment that is not a hybridization signal. In b, anti-β4-integrin co-labelling is in green and miR-203 in situ pseudocoloured signal is in red. c, qRT–PCR of FACS-purified cells from E15.5 epidermis reveals 25-fold more miR-203 in suprabasal (SB) versus basal layer (BL) cells (*P < 0.002). Error bars (s.d.) are derived from three experiments with basal layer level set as 1. Scale bars are 30 μm.

Figure 2

Figure 2. Premature activation of miR-203 in epidermis restricts its proliferative potential

a, In situ hybridization detects precociously expressed miR-203 in basal epidermis and hair germs of K14-miR-203 Tg skin. WT, wild type. b, By P0, Tg epidermis is thinner than the wild type. c–f, Signs of basal cell depletion in Tg skin. Arrows denote keratin-10 (K10)-positive, keratin-5 (K5)-negative, β4-integrin-low cells aberrantly juxtaposed to basement membrane. Note marked reduction of p63 in e. In f, quantifications reflect *P < 0.003, n = 8. g, Marked reduction in Tg versus wild-type colony-forming efficiency in vitro (**P < 0.0001, n = 3). In f and g, error bars (s.d.) are derived from eight or three experiments as indicated. h, Transduction of primary mouse keratinocytes with miR-203, but not empty vector or mutant miR-203M, results in a marked decline in S-phase cells that incorporate BrdU (*P < 0.001, **P < 0.0001). Values represent mean ± s.d. from three experiments. Scale bars are 30 μm.

Figure 3

Figure 3. Inhibition of miR-203 results in increased epidermal proliferation

a–d, P4 skins analysed after treatment of mice with mm-antagomir-203 (Ctrl) or antagomir-203 (anta). a, miR-203 in situ hybridizations. b–d, Immunofluorescence microscopy. Arrows denote basal-like features in suprabasal cells of antagomir-203-treated epidermis. PH3, phospho-histone H3. e, Elevated epidermal proliferation determined by BrdU injections at 4-h intervals and FACS quantification. f, The reduction in cycling cells that typically occurs upon calcium-induced differentiation of primary mouse keratinocytes in vitro is partially abrogated by anti-miR-203 oligonucleotides. Values represent mean ± s.d. from three experiments (*P < 0.005, **P < 0.0001). g, h, Epidermal defects that arise 30 days after tamoxifen-induced Dicer1 ablation (iKO). Arrows denote suprabasal p63. Scale bars are 30 μm.

Figure 4

Figure 4. MiR-203 targets p63 mRNA at 3′UTR

a, p63 protein is markedly diminished in Tg versus wild-type primary mouse keratinocytes. b, Immunoblot and qRT–PCR quantification of p63 expression. Top panel, calcium-induced (HiCa) p63 downregulation is largely relieved by anti-miR-203 oligonucleotides. Bottom panel, miR-203 transduction is sufficient to repress p63 protein expression in low calcium (LoCa). Right-hand graph, anti-miR-203 treatment slightly recovers p63 mRNA whereas miR-203 transduction marginally reduces p63 mRNA (*P < 0.01, n = 3). c, miR-203 transduction in LoCa primary mouse keratinocytes elevates p21 mRNA level (**P < 0.005, *P < 0.002, n = 3). d, Insertion of two miR-203 target sequences within mouse p63 3′UTR leads to diminished luciferase (Luc) reporter activity in the presence of miR-203 (*P < 0.002, n = 3). pGL3 is a luciferase-expressing plasmid from Promega. e, Mutation of both target sites (p63M2) abolishes miR-203-mediated repression of luciferase activity; a single-site mutation (p63M) has marginal effects (**P < 0.0001, n = 6). In HiCa, wild-type but not mutant p63 3′UTR represses luciferase activity (*P < 0.002, n = 3). For all graphs, error bars (s.d.) are derived from the number of experiments as indicated.

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