AUTHOR=He Tao , Fan Yu , Wang Yao , Liu Min , Zhu Alan Jian 

TITLE=Dissection of the microRNA Network Regulating Hedgehog Signaling in Drosophila

JOURNAL=Frontiers in Cell and Developmental Biology

VOLUME=10

YEAR=2022

URL=https://www.frontiersin.org/journals/cell-and-developmental-biology/articles/10.3389/fcell.2022.866491

DOI=10.3389/fcell.2022.866491

ISSN=2296-634X

ABSTRACT=<p>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 <italic>in vivo</italic> screening or <italic>in vitro</italic> assays. As miRNAs preferentially reduce target gene expression <italic>via</italic> 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 <italic>Drosophila</italic> toolbox of <italic>in vivo</italic> miRNA sensors for core components of Hh signaling, including <italic>hh</italic>, <italic>patched</italic> (<italic>ptc</italic>), <italic>smoothened</italic> (<italic>smo</italic>), <italic>costal 2</italic> (<italic>cos2</italic>), <italic>fused</italic> (<italic>fu</italic>), <italic>Suppressor of fused</italic> (<italic>Su(fu)</italic>), and <italic>cubitus interruptus</italic> (<italic>ci</italic>). With these tools in hand, we performed a genome-wide <italic>in vivo</italic> miRNA overexpression screen in the developing <italic>Drosophila</italic> wing imaginal disc. Of the twelve miRNAs identified, seven were not previously reported in the <italic>in vivo</italic> 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 <italic>miR-10</italic> and <italic>miR-958</italic> directly target <italic>fu</italic> and <italic>smo</italic>, 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 <italic>miR-10</italic> and <italic>miR-958</italic> target <italic>fu</italic> and <italic>smo</italic>, 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 <italic>in vivo</italic>. Given the powerful effects of these miRNAs on Hh signaling, we believe that identifying their <italic>bona fide</italic> targets of the other five miRNAs will help reveal important new players in the Hh regulatory network.</p>