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...

Egr3 is a nerve growth factor (NGF)-induced transcriptional regulator that is essential for normal sympathetic nervous system development. Mice lacking Egr3 in the germline have sympathetic target tissue innervation abnormalities and physiologic sympathetic dysfunction similar to humans with dysautonomia. However, since Egr3 is widely expressed and has pleiotropic function, it has not been clear whether it has a role within sympathetic neurons and if so, what target genes it regulates to facilitate target tissue innervation. Here, we show that Egr3 expression within sympathetic neurons is required for their normal innervation since isolated sympathetic neurons lacking Egr3 have neurite outgrowth abnormalities when treated with NGF and mice with sympathetic neuron-restricted Egr3 ablation have target tissue innervation abnormalities similar to mice lacking Egr3 in all tissues. Microarray analysis performed on sympathetic neurons identified many target genes deregulated in the absence of Egr3, with some of the most significantly deregulated genes having roles in axonogenesis, dendritogenesis, and axon guidance. Using a novel genetic technique to visualize axons and dendrites in a subpopulation of randomly labeled sympathetic neurons, we found that Egr3 has an essential role in regulating sympathetic neuron dendrite morphology and terminal axon branching, but not in regulating sympathetic axon guidance to their targets. Together, these results indicate that Egr3 has a sympathetic neuron autonomous role in sympathetic nervous system development that involves modulating downstream target genes affecting the outgrowth and branching of sympathetic neuron dendrites and axons.