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The location of follicular and epidermal stem cells in mammalian skin is a crucial issue in cutaneous biology. We demonstrate that hair follicular stem cells, located in the bulge region, can give rise to several cell types of the hair follicle as well as upper follicular cells. Moreover, we devised a double-label technique to show that upper follicular keratinocytes emigrate into the epidermis in normal newborn mouse skin, and in adult mouse skin in response to a penetrating wound. These findings indicate that the hair follicle represents a major repository of keratinocyte stem cells in mouse skin, and that follicular bulge stem cells are potentially bipotent as they can give rise to not only the hair follicle, but also the epidermis.

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M icroRNAs (miRNAs) are small, 20-to 24-nucleotide, noncoding RNAs found in diverse organisms. In animals, most miRNAs mediate posttranscriptional silencing by binding with partial complementarity to the 3Ј UTR of the target mRNA (1, 2). These endogenous, silencing RNAs have been shown to play important roles in development and differentiation (3-6), cellular stress responses (7), and cancer (8-11).The role of miRNAs in stratified squamous epithelia remains poorly understood. Inactivation of Dicer in mouse skin caused hair follicles to evaginate into the epidermis rather than invaginating downward, thus forming cyst-like structures (12, 13). These results underscore the importance of miRNAs in the regulation of epidermal and follicular development. miRNAs have also been extensively profiled in the corneal epithelium and show expression patterns that are regionally restricted (14). For example, miR-184 was the most abundant miRNA in the corneal epithelium; however, it was conspicuously absent from the limbal epithelium, an area enriched in corneal epithelial stem cells (15)(16)(17)(18). In contrast, miR-205 is broadly expressed throughout all viable cell layers in nearly all stratified squamous epithelia including the corneal, limbal, and conjunctival epithelia of the eye (12, 14). Thus, the corneal epithelium is unique in that it exhibits distinct as well as overlapping expression of miR-184 and miR-205 (14).miRNAs have been predicted to regulate thousands of mammalian genes (19); however, few targets have been experimentally validated for the great majority of these miRNAs. With the exception of a recent demonstration that a p63-related family member is negatively regulated by miR-203 (20), little is known about stratified squamous epithelial miRNA targets. We report that miR-205 represses SH2-containing phosphoinositide 5Ј-phosphatase 2 (SHIP2). Our finding that miR-184 negatively modulates the activity of miR-205 to maintain SHIP2 levels is the first demonstration that a miRNA can interfere with another to ensure the expression of a target protein. We show (i) that SHIP2 levels can be modulated in a variety of epithelial cells using gain-and loss-of-function experiments with miR-184 and miR-205 and (ii) that manipulating SHIP2 levels through miRNAs diminishes Akt signaling leading to decreased keratinocyte survival. Finally, we find a reciprocal relationship between miR-205 and SHIP2 expression in squamous cell carcinoma (SCC) cell lines and suggest that miR-205 may be viewed as a tumor promoter in the context of SCCs.Results miR-205 Targets SHIP2. We found miR-205 in all squamous epithelium that we examined (14). We also reported that miR-184 and miR-205 are the most abundant miRNAs in corneal epithelium and that miR-184 expression was restricted to the corneal epithelium (14). Bioinformatic analysis suggested that, in humans, the SHIP2 (Inppl1) 3Ј UTR is a putative target of both miR-184 and miR-205 (21) and is the only gene with overlapping binding sites to these two miRNAs. To test this prediction (Fig. 1...

Proper regulation of keratinocyte differentiation within the epidermis and follicular epithelium is essential for maintenance of epidermal barrier function and hair growth. The signaling intermediates that regulate the morphological and genetic changes associated with epidermal and follicular differentiation remain poorly understood. We tested the hypothesis that reactive oxygen species (ROS) generated by mitochondria are an important regulator of epidermal differentiation by generating mice with a keratinocyte-specific deficiency in mitochondrial transcription factor A (TFAM), which is required for the transcription of mitochondrial genes encoding electron transport chain subunits. Ablation of TFAM in keratinocytes impaired epidermal differentiation and hair follicle growth and resulted in death 2 weeks after birth. TFAM-deficient keratinocytes failed to generate mitochondria-derived ROS, a deficiency that prevented the transmission of Notch and β-catenin signals essential for epidermal differentiation and hair follicle development, respectively. In vitro keratinocyte differentiation was inhibited in the presence of antioxidants, and the decreased differentiation marker abundance in TFAM-deficient keratinocytes was partly rescued by application of exogenous hydrogen peroxide. These findings indicate that mitochondria-generated ROS are critical mediators of cellular differentiation and tissue morphogenesis.

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