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  • Although due to limitations of the current tools and technic

    2018-11-02

    Although, due to limitations of the current tools and technical challenges, we could not perform direct manipulation of the 11 miRNAs to rescue/inhibit neurogenesis in vivo, we demonstrated that most of these miRNAs are expressed in adult-born hippocampal neurons, and consequently depleted upon the loss of Dicer in vivo. Moreover, our in vitro experiments identify that all of the 11 miRNAs are sufficient to rescue DICER-dependent impairment of neurogenesis (Figure 6) and are required to sustain neurogenesis in WT aNSCs (Figure 7). Together, these results strongly indicate that miRNAs, rather than other additional DICER functions, determine neuronal fate of aNSCs. In perspective, our approach might be a useful paradigm to functionally investigate other miRNAs and targets. For example, 6 of the 11 miRNAs of our study are encoded by the Dlk1-Dio3 imprinted genomic region, containing the mirG locus that is highly enriched with miRNAs and deregulated in neurodevelopmental disorders and tyrosine kinase receptor tumors (Gardiner et al., 2012; Henriksen et al., 2014). This locus also encodes miR-134, which is important for neuronal synaptogenesis and plasticity (Schratt et al., 2006). The proposed synergy of miRNA actions refers to the “convergence” or “cooperativity” of miRNAs as a rapidly emerging theme in neurobiology, and has recently been proposed for embryonic neurogenesis (Barca-Mayo and De Pietri Tonelli, 2014), the adult SVZ (Santos et al., 2016), and apoptosis in the adult DG (Schouten et al., 2015). Consistent with this idea, we identified 26 putative targets of the 11 miRNAs that did not share immediate involvement in any pathway, but synergistically regulate biological processes such as neurogenesis, nervous system development, and neuronal differentiation. These results are consistent with our model whereby miRNAs “converge on function” (Barca-Mayo and De Pietri Tonelli, 2014). Given that each of the 11 miRNAs individually did not significantly induce neurogenesis, the synergic action on several targets from different pathways in parallel might compensate for the mild degree of miRNA-dependent regulation of individual mRNA targets. Further studies will be essential to experimentally validate the miRNAs and targets that are, in combination, key in regulating aNSCs neurogenesis. Finally, the identification of a set of miRNAs that determines neuronal fate of aNSCs raises interesting perspectives with regard to age-dependent loss of hippocampal neurogenesis (Marlatt and Lucassen, 2010) or the generation of undesirable cells upon insults or cell transplantation (Dibajnia and Morshead, 2013; Doetsch et al., 2002; Shimada et al., 2012; Sierra et al., 2015). Perhaps administration of the miRNAs that were the subject of this study will increase our repertoire of approaches to sustain neurogenesis in the aging brain, or to improve efficiency of NSC-based regenerative therapies.
    Experimental Procedures
    Author Contributions
    Acknowledgments We thank Dr. G. Hannon (Cold Spring Harbor Laboratory, USA) for kindly providing Dicer-Flox mouse line; Dr. S. Jessberger (University of Zurich, Switzerland) for Ascl1-ERT2-IRES-GFP viral construct; and Drs M. Götz and J. Ninkovic (Helmholtz Zentrum, München, Germany) for the split-Cre viral constructs. We thank Drs. P. Oloth (DZNE), T. Walker (CRTD), A. Simi, and A Contestabile (IIT) for advice on aNSC preparation and differentiation. We thank R. Pelizzoli and IIT-NBT technical staff (M. Pesce, F. Succol, and M. Nanni) for excellent help. We also thank the Animal Facility of IIT Genoa (F. Piccardi; D. Cantatore; R. Navone, and M. Morini) for assistance in animal experiments. D.D.P.T. was supported by intramural funds of Fondazione Istituto Italiano di Tecnologia. This research was supported by intramural funds of Fondazione Istituto Italiano di Tecnologia and by Fondazione Cariplo grant no. 2015-0590 to D.D.P.T. and F.N.
    Introduction Mouse embryonic stem cells (ESCs) from early-stage embryos have indefinite self-renewal capacity and can differentiate into cell types derivative of all three germ layers through asymmetric cell division (Niwa, 2007). Asymmetric cell division is a complex process whereby transcription, cell differentiation, cell cycle, and cell polarity must be coordinated in time and space (Noatynska et al., 2013). In vitro embryoid bodies (EBs) are powerful tools with which to study and understand the molecular mechanisms that underlie this process, as they mimic the in vivo developmental stages of peri-implantation embryos from epiblast to egg cylinder stages. An early step in this process is formation of the primitive endoderm (PE), an outer layer of polarized cells, followed by epiblast and primitive ectoderm development (Niwa, 2010).