Dissection of the microRNA Network Regulating Hedgehog Signaling in .

The evolutionarily conserved Hedgehog (Hh) signaling plays a critical role in embryogenesis and adult tissue homeostasis. Aberrant Hh signaling often leads to various forms of developmental anomalies and cancer. Since altered microRNA (miRNA) expression is associated with developmental defects and tumorigenesis, it is not surprising that several miRNAs have been found to regulate Hh signaling. However, these miRNAs are mainly identified through small-scale screening or assays. As miRNAs preferentially reduce target gene expression the 3' untranslated region, we analyzed the effect of reduced expression of core components of the Hh signaling cascade on downstream signaling activity, and generated a transgenic toolbox of miRNA sensors for core components of Hh signaling, including , (), (), (), (), (), and (). With these tools in hand, we performed a genome-wide miRNA overexpression screen in the developing wing imaginal disc. Of the twelve miRNAs identified, seven were not previously reported in the Hh regulatory network. Moreover, these miRNAs may act as general regulators of Hh signaling, as their overexpression disrupts Hh signaling-mediated cyst stem cell maintenance during spermatogenesis. To identify direct targets of these newly discovered miRNAs, we used the miRNA sensor toolbox to show that and directly target and , respectively, while the other five miRNAs act through yet-to-be-identified targets other than the seven core components of Hh signaling described above. Importantly, through loss-of-function analysis, we found that endogenous and target and , respectively, whereas deletion of the other five miRNAs leads to altered expression of Hh signaling components, suggesting that these seven newly discovered miRNAs regulate Hh signaling . Given the powerful effects of these miRNAs on Hh signaling, we believe that identifying their targets of the other five miRNAs will help reveal important new players in the Hh regulatory network.