By conditional ablation of Dicer specifically

By conditional ablation of Dicer specifically in bona fide aNSCs of the adult hippocampus in vivo and in vitro and by manipulation of specific miRNAs, here we studied the role of miRNAs for lineage fate choice of aNSCs. Our study identified a set of 11 miRNAs that, by synergistic enforcement of gene-regulatory networks, allows aNSCs to acquire the neurogenic fate at the expense of astrogliogenesis.
Results
Discussion Our study demonstrates that pak1 a set of 11 miRNAs (of which nine were not previously characterized in adult neurogenesis) is essential for neurogenic lineage fate determination of aNSCs in the adult hippocampus, and does so at the expense of astrogliogenesis. Remarkably, these miRNAs could rescue impaired neurogenesis in Dicer cKO aNSCs to WT levels only when administered as a pool, not individually. Thus our study provides evidence for the emerging notion of miRNA “convergence” (or “cooperativity”) that, by synergistic enforcement of gene-regulatory networks, allows the acquisition of neurogenic fate programming in aNSCs. Adult neurogenesis is a highly conserved process among vertebrates (Gage and Temple, 2013). However, the mechanisms underlying the control of a proper acquisition of the neurogenic versus astrogliogenic fate remains a fundamental question in the field (Bonaguidi et al., 2012; Kempermann, 2011). Here, by targeting bona fide type I aNSCs in vivo and in vitro, we show that loss of DICER-dependent miRNAs in aNSCs impaired neurogenesis but not astrogliogenesis. Thus our results uncover miRNAs as a regulatory level necessary to sustain neurogenic lineage and prevent astrogliogenesis in the adult hippocampal niche. This evidence reinforces the emerging idea that multiple layers of control are required to allow adult neurogenesis to occur properly. This has recently been demonstrated for other epigenetic mechanisms in a similar way (Lv et al., 2013; Noguchi et al., 2015). An interesting question is why, given these results, DICER/miRNA-depleted aNSCs can still undergo astrocytic differentiation at all? Based on our results, and given the proposed glial nature of aNSCs (Brunne et al., 2010; Kriegstein and Alvarez-Buylla, 2009; Nicola et al., 2015), which share common molecular pathways with non-neurogenic astrocytes (Beckervordersandforth et al., 2014; Buffo et al., 2008; Coskun et al., 2008), we postulate that the gliogenic program in aNSCs might represent rather a “default” developmental path than a fate change, and thus be less dependent on miRNAs (Encinas et al., 2011). Despite we cannot rule out that “immature or intermediate” astrocytes are generated by Dicer-depleted aNSCs in vivo, these newborn cells (as revealed by Td-Tomato and BrdU) were also positive for different astrocytic markers, such as S100b (Figures 1E and 1H) and GS (Figures S1B and S1C) 2 months after Dicer deletion. Moreover, since upon Dicer ablation we did not find increased expression of progenitor markers such as Nestin or SOX2 (Figures S1B and S1C), we postulate that these cells might be bona fide astrocytes, rather than aNSCs remaining in undifferentiated (or returning) or quiescent state. Another possibility is that miRNA-depleted newborn neurons could be more susceptible to apoptosis compared with astrocytes. However, differentiating aNSC Dicer HT (Figure 2) were not more susceptible than WT cells to apoptosis (Figure S4), but still gave rise to fewer neurons (Figure 3). This suggests that miRNA loss in aNSCs can affect the switch toward neurogenesis independently from cell death. Finally, it is still possible that different subtypes of neural and glial progenitor cells exist in the adult hippocampal niche that responds differently to miRNA depletion; hence in the absence of Dicer/miRNAs neurogenesis fails while astrogliogenesis is proportionally increased. This scenario would be consistent with the known heterogeneity of aNSCs (Shin et al., 2015). However, we can still conclude that loss of Dicer and DICER-dependent miRNAs does not impair astrogliogenesis in the adult hippocampal stem cell niche.