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MicroRNA-205 controls neonatal expansion of skin stem cells by modulating the PI(3)K pathway

Nature Cell Biology volume 15, pages 1153–1163 (2013)Cite this article

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Abstract

Skin stem cells (SCs) are specified and rapidly expanded to fuel body growth during early development. However, the molecular mechanisms that govern the amplification of skin SCs remain unclear. Here we report an essential role for miR-205, one of the most highly expressed microRNAs in skin SCs, in promoting neonatal expansion of these cells. Unlike most mammalian miRNAs, genetic deletion of miR-205 causes neonatal lethality with severely compromised epidermal and hair follicle growth. In the miR-205 knockout skin SCs, phospho-Akt is significantly downregulated, and the SCs prematurely exit the cell cycle. In the hair follicle, this accelerates the transition of the neonatal SCs towards quiescence. We identify multiple miR-205-targeted negative regulators of PI(3)K signalling that mediate the repression of phospho-Akt and restrict the proliferation of SCs. Our findings reveal an essential role for miR-205 in maintaining the expansion of skin SCs by antagonizing negative regulators of PI(3)K signalling.

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Figure 1: miR-205 is highly expressed in skin progenitors and SCs.
Figure 2: Generation of miR-205 KO mice.
Figure 3: Genetic deletion of miR-205 causes neonatal lethality with severe skin defects.
Figure 4: Ablation of miR-205 compromises the proliferation of interfollicular progenitors.
Figure 5: miR-205 is required for the expansion of HFSCs during neonatal development.
Figure 6: Ablation of miR-205 impairs the proliferation of K5+ basal cells in stratified epithelium.
Figure 7: miR-205 directly targets multiple negative regulators of the PI(3)K/Akt pathway.
Figure 8: miR-205 is required to maintain pAkt levels by targeting multiple negative regulators in skin progenitors and SCs.

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Acknowledgements

We are grateful to E. Fuchs for K14–RFP mice. We thank T. Blumenthal, T. Cech, B. Cullen, M. Han, M. Winey and X-J. Wang for comments on the manuscript. We thank C. Yang, J. Gao and D. Feng for the generation of miR-205 KO; S. Ha and L. Greiner for assistance in the animal facility; Y. Han for FACS; and G. Voeltz for confocal microscopy. We also thank members of the Yi laboratory for their critical discussions. This publication was made possible by a start-up fund provided by the University of Colorado and Grant Number R00AR054704 and R01AR059697 (R.Y.) and R01GM083300 (E.C.L.).

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Author notes

  1. Evan O’Loughlin

    Present address: Present address: Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, Massachusetts 02115, USA,

  2. Zhaojie Zhang and Evan O’Loughlin: These authors contributed equally to this work

Authors and Affiliations

  1. Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309, USA

    Dongmei Wang, Zhaojie Zhang, Evan O’Loughlin, Li Wang, Xiying Fan & Rui Yi

  2. Sloan-Kettering Institute, 1017 Rockefeller Research Labs, 1275 York Avenue Box 252, New York, New York 10065, USA

    Eric C. Lai

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Contributions

R.Y. conceived the study. D.W. carried out most experiments and analysed the data with assistance from Z.Z. (bioinformatic analysis), E.O. (in situ hybridization), L.W. (ChIP-seq and RNA-seq) and X.F. (target validation). E.C.L. provided critical resources. R.Y. and D.W. wrote the manuscript with input from all authors.

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Integrated supplementary information

Supplementary Figure 1 miRNAs are required for the expansion and maintenance of epidermal progenitor and hair follicle stem cells.

(a) At P4.5, the Dicer cKO pup is smaller and shows less developed hair coat and dehydrated skin, compared to the WT littermate. (b) At birth (P0.5), HFSCs are similarly specified and detected in both WT and Dicer cKO as indicated by Nfatc1 staining. (c and d) Loss of HFSCs in the bulge of Dicer cKO skin by P4.5. Nfatc1+ HFSCs are completely absent in Dicer cKO (c). Sox9+ HFSCs are also significantly reduced in the bulge (d). White dotted lines mark the epidermal/dermal boundary; brackets denote the bulge region. IFE, interfollicular epidermis; Epi, epidermis; ORS, outer root sheath; Mx, matrix; Bu, bulge. Scale bars, 20 μm.

Supplementary Figure 2 Expression pattern of miR-205 in adult skin and FACS purification strategy to specifically isolate HFSCs using K14-RFP/Sox9-GFP mice.

(a) By in situ hybridization, expression of miR-205 is universally high in telogen adult skin, while its expression in anagen adult skin shows the highest expression in the bulge region, mimic its expression pattern in neonatal stage. (b) miR-205 expresses at lower level in active anagen HFSCs comparing to quiescent telogen HFSCs. Lin−CD34+α6hi HFSCs were FACS purified from anagen and telogen HFs and miR-205 level was measured by qRT-PCR. Data shown are mean ± s.d. from 5 independent experiments. ***, P<0.001 by Student’s t-test. (c) FACS purification strategy to specifically isolate HFSCs using K14-RFP/Sox9-GFP mice. P4.5 total back skin is first separated into epidermal sheet and HF/dermis part by dispase treatment. One dissected K14-RFP/Sox9-GFP hair follicle is shown. All K14-RFP positive hair follicle cells are marked by red fluorescence, while hair follicle stem cells, which express Sox9, are marked by green fluorescence. HFSC is purified as K14RFPhi/Sox9GFP+/α6integrin+, ORS is sorted as K14RFPhi/Sox9GFP−/α6integrin+, matrix as K14RFPlow/Sox9GFP−. White dotted lines mark the epidermal/dermal boundary. Scale bars, 20 μm.

Supplementary Figure 3 miR-205 is expressed in organs with stratified epithelial tissue.

(a) miR-205 is co-expressed with K5 in stratified epithelial tissue. Using in situ hybridization, miR-205 is detected in the epithelial tissue of skin, tongue, oesophagus, stomach and bladder where K5 is expressed. White dotted lines mark the epidermal/dermal boundary. (b) Most vital organs are negative for miR-205. Expression of miR-205 or K5 is not detected in intestine, heart, lung, brown fat, spleen, liver or kidney by in situ hybridization and immunofluorescence staining. Scale bars, 20 μm.

Supplementary Figure 4 miR-205 is conserved in vertebrates.

(a) The only highly conserved region in the primary transcript of miR-205 is the miR-205 hairpin. (b) The mature sequences of miR-205 are conserved in vertebrates but not in lower eukaryotes e.g. worm and fly.

Supplementary Figure 5 Terminal differentiation in both epidermal and hair follicle lineages is not altered in miR-205 KO.

(a) K5 marks the basal layer; K1 marks the spinous layer. (b) Loricrin (Lori) marks the granular layer. (c) AE13 marks the hair shaft. White asterisks denote autofluoscence of stratum corneum; white dotted lines mark the epidermal/dermal boundary. Scale bars are 50 μm.

Supplementary Figure 6 Full-thickness grafting. Back skin from P0.5 littermates of WT and KO was grafted onto nude mice.

(a) Images show the skin right after putting the donor skin onto the host nude mouse. (b) Bandage was removed at 14 days after grafting. At day 15, hair shaft could be seen from the grafted skin and the grafts were taken for analysis.

Supplementary Figure 7 Characterization of the transition from proliferation to quiescence of bulge stem cells in early stage HFs.

(a and b) In E17.5 hair germ (a) and P0.5 rudimentary HFs (b), all the Nfatc1+ bulge stem cells are positive for Ki67, indicating they are actively proliferating. (c) In as early as P1.5 HF, some Nfatc1 cells have already exited cell cycle, become quiescent, marked by Ki67 negative staining. White dotted lines mark the epidermal/dermal boundary; arrowheads indicate Ki67 negative/Nfatc1 positive bulge stem cells in P1.5 HF. Scale bars, 20 μm.

Supplementary Figure 8 miR-205 directly regulates negative regulators of the PI3K/Akt pathway.

(a-c) The scan for 7mer motifs reveals the enrichment of miR-205 seed (1-7, 2-8 and 3-9) matches in the 3′ UTRs of upregulated genes. All 7mer matches to the miR-205 sequences (1-22nt) are determined in the 3′ UTR, 5′ UTR and coding sequences (CDS) of three gene categories: genes that are upregulated more than 20% in the miR-205 KO HFSCs; genes that are downregulated more than 20% in the miR-205 KO HFSCs; and all genes. The percentage of genes from each category that contains any given 7mer match is calculated and plotted in the Y-axis. Note that only 1-7, 2-8 and 3-9 motifs show enrichment in the 3′UTR of the upregulated genes. (d) The same scan for 7mer motifs of miR-1 in the upregulated genes fails to show any enrichment, indicating the specificity of the upregulated genes for miR-205. (e) Base-pairing between the miR-205 seed (red) and its target sites in the 3′ UTR of candidate genes and the mutated target sites (green). (f) Phospho-Akt level is not affected by miR-205 KO in other vital organs. Numbers between panels represent the densitometry values of phosphorylated protein normalized to total levels. (g) Inhibition of the PI3K pathway abolishes the growth of keratinocytes in colony formation assay. Primary keratinocytes were treated with DMSO (control) or the PI3K small molecule inhibitor LY294002. The treatment of LY294002 completely abolishes cell growth.

Supplementary Figure 9 Uncropped images of immunoblots.

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Wang, D., Zhang, Z., O’Loughlin, E. et al. MicroRNA-205 controls neonatal expansion of skin stem cells by modulating the PI(3)K pathway. Nat Cell Biol 15, 1153–1163 (2013). https://doi.org/10.1038/ncb2827

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