br Materials and methods br Results br

Materials and methods
Results
Discussion We have identified and begun to characterize a novel miRNA, miR-410, which on inhibition of BMP signaling by Noggin, is down-regulated in the adult SVZ. Over-expression of miR-410 in SVZ neurospheres inhibited neuronal differentiation and increased the number of astrocytes produced. Loss of function of miR-410 had the opposite effect – promoting neuronal differentiation at the expense of astrocyte formation, while co-expression of miR-410 with Noggin reduced the increase in neuronal differentiation caused by Noggin to control levels, suggesting that miR-410 functions downstream of BMP signaling. To understand the mechanisms underlying these effects, we tested multiple candidate targets of miR-410: Elavl4, Sox1, Smad7, Tcf4, and Fgf7, which were down-regulated by miR-410. In fact, co-expression of miR-410 resistant Elavl4 also reversed the decrease in neuronal differentiation caused by miR-410, suggesting Elavl4 acts downstream of miR-410. We also observed that while over-expression of Elavl4 without the 3′UTR drastically increased neuronal differentiation, over-expression of Elavl4 with either the full-length or truncated 3′UTR containing the miR-410 site did not alter neuronal differentiation. This suggests that endogenous miR-410, and other miRNAs that target the short truncated 3′UTR are sufficient to abrogate the pro-neuronal differentiation function of Elavl4. Surprisingly, we also observed that over-expression of miR-410 had an impact on neuronal morphology, with miR-410 over-expressing neurons characterized by processes that were short and less branched–which may be a product of Elavl4\'s ability to regulate neurite formation (Abdelmohsen et al., 2010). Interestingly, one downstream myd88 pathway of the miR-379-410 cluster is Pumilio2 (Fiore et al., 2009), an important regulator of dendritogenesis, so that miR-410 could also affect Pumilio2 proteins to regulate process outgrowth. Although the functions of miRNAs in neurogenesis have been widely studied (De Pietri Tonelli et al., 2008; Cheng et al., 2009; Zhao et al., 2009; Szulwach et al., 2010), there is little information regarding crosstalk between exogenous growth factors and miRNA expression or function (Terao et al., 2011; Wang et al., 2012; Kao et al., 2013). Here we demonstrate that the BMP signaling pathway plays a role in NSC lineage differentiation by regulating miR-410 and its target genes. miR-410 is encoded in the miRNA containing gene Mirg, a member of the maternally imprinted Dlk2-Gtl2 gene cluster, which is enriched in the brain (Tierling et al., 2006; Han et al., 2012). We have identified multiple BMP/Smad responsive elements in the enhancer region of the Mirg gene. Interestingly, expression of two other miRNAs from the same cluster, miR-382 and miR-377, are regulated by TGF-β signaling (Lan and Chung, 2011; Milosevic et al., 2012), suggesting that Smad signaling may play a crucial role in the regulation of the entire miRNA cluster. In addition, Smad 1 and 5 have been reported to bind the pri-miR-21 hairpin and promote miR-21 biogenesis (Davis et al., 2008; Blahna and Hata, 2012). BMP signaling and downstream Smad proteins may similarly regulate the expression of miR-410 and it will be of interest to determine if BMP-specific Smad 1/5/8 signaling directly regulates the expression of Mirg and miR-410. While the astrocyte is predominant fate of adult SVZ neurospheres (Li et al., 2010), few miRNAs have been shown to regulate astrocyte differentiation (Zhang et al., 2013), unlike the many miRNAs that have been reported to regulate neuronal cell fate (Cheng et al., 2009; Aranha et al., 2011). BMP signaling also plays an important role in oligodendrocyte differentiation, as interfering with Smad signal transduction (Colak et al., 2008) or application of another BMP inhibitor, Chordin, can redirect neuronal precursors to an oligodendrocyte fate (Jablonska et al., 2010). We have observed that miR-410 over-expression leads to a reduction in OPC numbers; however, miR-410 loss of function did not significantly affect OPC differentiation. This suggests that miR-410 may inhibit oligodendrocyte lineage commitment through a second mechanism, and/or there are additional controls on OPC differentiation.