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Mef2-mediated transcription of the miR379–410 cluster regulates activity-dependent dendritogenesis by fine-tuning Pumilio2 protein levels

2009; Springer Nature; Volume: 28; Issue: 6 Linguagem: Inglês

10.1038/emboj.2009.10

1460-2075

Roberto Fiore, Sharof Khudayberdiev, Mette Christensen, Gabriele Siegel, Steven W. Flavell, Tae-Kyung Kim, Michael E. Greenberg, Gerhard Schratt,

Circular RNAs in diseases

Article5 February 2009Open Access Mef2-mediated transcription of the miR379–410 cluster regulates activity-dependent dendritogenesis by fine-tuning Pumilio2 protein levels Roberto Fiore Roberto Fiore Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Sharof Khudayberdiev Sharof Khudayberdiev Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Mette Christensen Mette Christensen Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Wilhelm Johannsen Center for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej, Denmark Search for more papers by this author Gabriele Siegel Gabriele Siegel Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Steven W Flavell Steven W Flavell Department of Neurobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Tae-Kyung Kim Tae-Kyung Kim Department of Neurobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Michael E Greenberg Michael E Greenberg Department of Neurobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Gerhard Schratt Corresponding Author Gerhard Schratt Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Roberto Fiore Roberto Fiore Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Sharof Khudayberdiev Sharof Khudayberdiev Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Mette Christensen Mette Christensen Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Wilhelm Johannsen Center for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej, Denmark Search for more papers by this author Gabriele Siegel Gabriele Siegel Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Steven W Flavell Steven W Flavell Department of Neurobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Tae-Kyung Kim Tae-Kyung Kim Department of Neurobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Michael E Greenberg Michael E Greenberg Department of Neurobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Gerhard Schratt Corresponding Author Gerhard Schratt Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany Search for more papers by this author Author Information Roberto Fiore1,‡, Sharof Khudayberdiev1,‡, Mette Christensen1,2, Gabriele Siegel1, Steven W Flavell3, Tae-Kyung Kim3, Michael E Greenberg3 and Gerhard Schratt 1 1Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, Universität Heidelberg, and Institut für Neuroanatomie, Universitätsklinikum Heidelberg, Heidelberg, Germany 2Wilhelm Johannsen Center for Functional Genome Research, Department of Cellular and Molecular Medicine, University of Copenhagen, Blegdamsvej, Denmark 3Department of Neurobiology, Harvard Medical School, Boston, MA, USA ‡These authors contributed equally to this work *Corresponding author. Interdisziplinäres Zentrum für Neurowissenschaften, SFB488 Junior Group, University of Heidelberg, Im Neuenheimer Feld 345, 69210 Heidelberg, Germany. Tel.: +49 6221 566210; Fax: +49 6221 567897; E-mail: [email protected] The EMBO Journal (2009)28:697-710https://doi.org/10.1038/emboj.2009.10 There is a Have you seen ...? (March 2009) associated with this Article. PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Neuronal activity orchestrates the proper development of the neuronal circuitry by regulating both transcriptional and post-transcriptional gene expression programmes. How these programmes are coordinated, however, is largely unknown. We found that the transcription of miR379–410, a large cluster of brain-specific microRNAs (miRNAs), is induced by increasing neuronal activity in primary rat neurons. Results from chromatin immunoprecipitation and luciferase reporter assays suggest that binding of the transcription factor myocyte enhancing factor 2 (Mef2) upstream of miR379–410 is necessary and sufficient for activity-dependent transcription of the cluster. Mef2-induced expression of at least three individual miRNAs of the miR379–410 cluster is required for activity-dependent dendritic outgrowth of hippocampal neurons. One of these miRNAs, the dendritic miR-134, promotes outgrowth by inhibiting translation of the mRNA encoding for the translational repressor Pumilio2. In summary, we have described a novel regulatory pathway that couples activity-dependent transcription to miRNA-dependent translational control of gene expression during neuronal development. Introduction The development and refinement of neuronal circuitry are regulated by both intrinsic and activity-dependent programmes of gene expression (Flavell and Greenberg, 2008). The latter is particularly important at later stages of neuronal development, such as dendritogenesis (Chen and Ghosh, 2005) and synapse formation (Waites et al, 2005). An important layer of regulation is new transcription induced by activity-regulated transcription factors (Hong et al, 2005). For example, CREB has a key function in activity-dependent dendritic outgrowth of central neurons (Lonze and Ginty, 2002; Redmond et al, 2002). On the other hand, the activity-dependent transcription factor, myocyte enhancing factor 2 (Mef2), functions as a negative regulator of excitatory synapse number (Flavell et al, 2006; Shalizi et al, 2006). Morphological abnormalities in dendritic arbourization and postsynaptic structure are common hallmarks of a number of cognitive diseases, for example, mental retardation (Bagni and Greenough, 2005). Activity-dependent regulation of gene expression involves, in addition to the activation of new transcriptional programmes within the nucleus, also post-transcriptional control of pre-existing mRNAs (Steward, 2002). The expression of pre-existing mRNAs is often regulated locally in dendrites close to synaptic contacts (Sutton and Schuman, 2006). Important post-transcriptional regulatory molecules are RNA-binding proteins (i.e. CPEB, Pumilio (Pum), etc.) that regulate transport, stability or translation of the mRNAs in response to activity (Kiebler and Bassell, 2006; Richter, 2007). MicroRNAs (miRNAs) are another class of key post-transcriptional regulators that can bind to the 3′UTR of target mRNAs to downregulate their expression by inducing either mRNA degradation or translational suppression (Kosik, 2006; Fiore et al, 2008). Little is known, however, on how these two mechanisms of activity-dependent gene expression, global transcriptional control and local post-transcriptional control, are coordinated within a neuron. We have previously shown that miR-134 controls spine morphogenesis in rat hippocampal neuron by repressing local translation of the LimK1 mRNA within dendrites (Schratt et al, 2006). BDNF, which is secreted in response to neuronal activity, relieves the translational block of the LimK1 mRNA thereby allowing spine growth. It is still an open question whether miR-134 function is also controlled globally within a neuron at the transcriptional level. Intriguingly, the miR-134 gene is clustered together with more than 50 other miRNAs within the Gtl2/Dlk1 locus (referred hereafter as the miR379–410 cluster) (Seitz et al, 2004). Clustered miRNA genes are often co-expressed, a prerequisite for the coordinated control of related biological processes (He et al, 2005). In this study, we provide evidence that the entire miR379–410 cluster is co-regulated at the transcriptional level by neuronal activity in a Mef2-dependent manner. Importantly, activity-dependent regulation of multiple miRNAs from the cluster, including miR-134, is necessary for the correct elaboration of the dendritic tree. Furthermore, we show that the RNA-binding protein Pum2 is a direct miR-134 target and a key mediator of the miR-134 growth-promoting effect on dendritogenesis. Our results point to the miR379–410 cluster, in particular miR-134, as a key component of a mechanism that couples transcriptional and local control of gene expression in response to neuronal activity during the development of neural circuitry. Results The miR379–410 cluster is co-regulated by neuronal activity Recently, we provided evidence that miR-134 regulates activity-dependent control of dendritic mRNA translation in response to BDNF. We decided to investigate whether neuronal activity also regulates miR-134 and possibly the entire miR379–410 cluster globally within the cell at the transcriptional level. We first tested whether expression of candidate miRNAs of the miR379–410 cluster was coordinately induced by neuronal activity. We used either membrane-depolarizing concentrations of KCl, which leads to Ca2+ influx, or the application of BDNF, a growth factor released by synaptic stimulation, as paradigms to mimic neuronal activity in a culture of dissociated neurons (Redmond et al, 2002; Wayman et al, 2006). Primary cortical neurons cultured for 5 days in vitro (5DIV) were treated with either BDNF or KCl for up to 6 h. After isolation of total RNA, the expression of pre-miRNAs that are encoded at different positions within the GTl2/DLK1 locus was analysed by quantitative RT–PCR (Figure 1A). Consistent with our earlier findings, miRNAs from the miR379–410 cluster (including miR-134) are expressed at very low levels in unstimulated neurons at this early developmental stage. Strikingly, all of the tested pre-miRNAs located within miR379–410 were robustly induced by both BDNF and KCl stimulation. Similar to the known activity-regulated cFos gene, miR379–410 pre-miRNA induction was both rapid and transient, peaking at 2 h and lasting for at least 6 h. The level of the neighbouring Gtl2 transcript was not affected by KCl and BDNF (Figure 1A–C), demonstrating that our treatment led to a specific induction of the miR379–410 domain. Figure 1.The miR379–410 cluster is co-regulated by neuronal activity. (A) Schematic representation of the mouse GTL2/RTL1 locus on distal chromosome 12. miRNA genes are indicated by triangles, small nucleolar RNAs (SnoRNA) by filled bars, the non-coding RTL1 and GTL2 genes by grey rectangles and the miR379–410 cluster by an open rectangle. Arrows point to miRNAs analysed by RT–PCR and sensor assays. Diagram is not drawn to scale. (B) Membrane depolarization increases miR379–410 precursor expression. Quantitative RT–PCR analysis of total RNA extracted from KCl-stimulated primary cortical neurons. DIV5 cortical neurons were treated for 6 h with 16 mM KCl, and total RNA was isolated at different time points during the stimulation period and analysed by real-time PCR with primers for different miRNA precursors located within the GTL2/RTL1 locus, cFos (positive control) and GTL2 (negative control). The data are normalized to β3-tubulin and presented as relative to the basal. Data represent the average of three independent experiments+s.d. cFos induction values are out of scale and indicated in the insert. (C) BDNF treatment increases miR379–410 precursor expression. Real-time PCR analysis of total RNA extracted from BDNF-stimulated primary cortical neurons. DIV5 cortical neurons were treated for 6 h with 40 ng/ml BDNF; total RNA was isolated at different time points during the stimulation and analysed as in (B). Data represent the average of three independent experiments+s.d. cFos induction values are out of scale and indicated in the insert. (D) Effect of membrane depolarization on the subcellular localization of miR-134 in hippocampal neurons. DIV7 rat hippocampal neurons were stimulated for 6 h with 16 mM KCl, fixed and analysed by fluorescent in situ hybridization. A DIG-labelled LNA probe directed against miR-134 (miR-134 probe) and a DIG-labelled control probe of unrelated sequence (mismatch probe) were used (5 pmol each). Representative images for unstimulated cells (left panels) and KCl-treated neurons (right panels) are shown. Higher panels show the robust increase in miR-134 signal in both the neuronal soma (asterisks) and dendrites (arrowheads) upon KCl stimulation. Scale bars: 20 and 5 μm. (E) Quantification of miR-134 levels obtained by ISH analysis. Ten pictures for each experimental condition were measured to calculate the average intensity of the fluorescent signal obtained with the indicated probes. Data are presented as the fold increase in average intensity in KCl-treated versus unstimulated whole cells (total) and dendrite only (dendritic). Error bars represent the average of two independent experiment+s.d. (F) Membrane depolarization increases functional miR379–410 miRNAs. An miRNA sensor assay was performed in KCl-stimulated hippocampal neurons. The principle of an miRNA sensor assay is described on the left. Right: hippocampal neurons (DIV4) were transfected with the indicated sensors (50 ng) alone or in combination with the indicated anti-miRs (50 nM). After KCl incubation (DIV7), cells were fixed and miRNA-positive cells were scored based on the fluorescent sensor signal. Results from one representative out of three independent experiments are shown. Download figure Download PowerPoint We next investigated the effect of membrane depolarization on the levels and subcellular localization of one of the miR379–410 miRNAs, miR-134, by in situ hybridization (ISH) of primary hippocampal neurons (DIV7). We used a probe that was able to recognize both mature and pre-miR-134. (Figure 1D). Low levels of miR-134 were detectable in unstimulated neurons and KCl led to a robust and specific increase in miR-134 ISH signal, confirming our results obtained with quantitative RT–PCR (Figure 1B). The KCl-mediated increase was completely abolished by pretreatment of neurons with actinomycin D, demonstrating that the increase was due to de novo miR-134 transcription (data not shown). The increase of miR-134 upon depolarization was not restricted to the soma but the miR-134 signal was also evident in dendrites (Figure 1D, higher magnification panels). Quantification of the in situ signal confirmed the depolarization-induced increase of miR-134 in both the somatic and dendritic compartments (Figure 1E). The robust increase of miR-134 in dendrites suggests a local function in this compartment. So far, our expression analysis did not directly address whether our activity paradigm induces functional miR379–410 miRNAs. To verify that neuronal activation induces the expression of mature and functional miRNAs from the cluster, we used a previously described single cell sensor assay (Mansfield et al, 2004). We used bicistronic GFP/dsRED expression vectors (‘sensor’), the expression of which is controlled by miR379–410 miRNAs due to two perfectly complementary miRNA-binding sites in the 3′UTR of the dsRED gene. Neurons that express functional miRNA (miRNA positive) are identified by the lack of dsRED due to RISC-mediated cleavage of the dsRED mRNA (Figure 1F). The number of miRNA-positive neurons increased upon depolarization for all tested miR379–410 miRNAs (Figure 1F), validating that neuronal activity induces functional miRNAs. Co-transfection of the sensors with specific 2’O-methyl antisense oligonucleotides (anti-miRs) confirmed that the increase in miRNA-positive cells was due to a specific elevation of miRNA activity. These results also demonstrate that anti-miRs can be used to specifically interfere with the function of the KCl-induced miRNAs miR-329, -134 and -541. Mef2 is necessary for activity-dependent regulation of the miRNA-379–410 cluster We devised a comparative genomic approach based on sequence conservation to identify regulatory elements that could mediate activity-dependent transcription of the miR379–410 cluster. Highly conserved regions within 20 kb upstream of the cluster were screened for potential binding sites for activity-regulated transcription factors (Figure 2A). Thereby, we identified 10 potential binding sites for Mef2, a known activity-regulated transcription factor that negatively regulates synapse number in mature hippocampal neurons (Shalizi and Bonni, 2005). The occupancy of the putative Mef2-binding sites (MBSs) in vivo was assessed by chromatin immunoprecipitation (ChIP) using DIV5 primary cortical neurons (Figure 2B and data not shown). A PCR product encompassing one of the potential MBS, MBS10, could be specifically amplified from immunoprecipitates of formaldehyde-fixed chromatin using a Mef2-specific antibody (Figure 2B, upper panel), in a similar manner as the known Mef2 target gene Nur77 (Figure 2B, upper and lower panels). In contrast, Mef2 antibodies were unable to enrich chromatin from the β-globin locus that lacks a MBS, confirming the specificity of our ChIP protocol. Therefore, Mef2 is bound to MBS10 in neurons in vivo. To begin to assess the relevance of MBS10 in activity-dependent transcription of miR379–410, we first monitored the presence of a transcript between the miR-379 gene and MBS10. Using RT–PCR with a set of overlapping primers located within the region from MBS10 to miR-379, we were able to detect a continuous transcript except for a small gap (Figure 2A and C, primer pairs 6 and 7, and data not shown). This gap consists of highly repetitive sequence that is likely resistant to PCR amplification. In further support of the existence of a long, continuous transcript spanning the region from MBS10 all the way to the end of the miR379–410 cluster, we found that the detected PCR fragments, similar to the adjacent miRNA genes, were robustly induced by depolarization (Figure 2C). Taken together, our transcript analysis supports the idea that the miR379–410 cluster is transcribed as a single polycistronic unit starting in the proximity of MBS10. Figure 2.A Mef2-binding site (MBS10) is located upstream of the miR379–410 cluster. (A) Alignment of the 20 kb directly upstream of the miR379–410 cluster. Peaks represent conserved regions, the position of MBS10 and the sequence conservation of the consensus are shown. The different PCR fragments that were amplified are indicated by black numbers, fragments that failed to be amplified due to the repetitive nature of the sequence are indicated by blue numbers. (B) MBS10 is bound by Mef2 in native chromatin in vivo. ChIP was performed in primary cortical neurons (5DIV) using an anti-Mef2 antibody or IgG as a control. Primers that specifically amplify the genomic region of MBS10, β-globin or Nur77 promoter were used for PCR amplification. Input corresponds to genomic DNA isolated before immunoprecipitation. One representative out of three independent experiments is shown. (C) A continuous transcript spans the region between MBS10 and miR-379. Semiquantitative RT–PCR analysis was performed on cortical neurons (5DIV) that were either left untreated or membrane depolarized (16 mM KCl, 6 h). Overlapping primer pairs specific for the genomic location indicated in (A) were used. Only the amplification products of a few selected PCR reactions are shown. Download figure Download PowerPoint To test the functionality of MBS10, we cloned either the wild-type or a mutated MBS10 upstream of a minimal promoter driving the firefly luciferase reporter gene (pGL3-MBS10). The expression of a constitutively active mutant of Mef2 (Mef2-VP16) in cortical neurons induced luciferase activity from MBS10 reporter construct (Figure 3A) to a similar extent as a promoter construct derived from a known Mef2 target gene (pGL3-Nur77). The DNA-binding deficient mutant Mef2ΔDBD-VP16 failed to increase the activity of both the Nur77 and MBS10 luciferase reporters, suggesting that induction is dependent upon Mef2 binding. This is further supported by the lack of induction of the pGL3-MBS10mut upon co-transfection with Mef2-VP16 (Figure 3A). We next investigated the role of endogenous Mef2 in activity-dependent transcriptional control of the cluster. KCl or BDNF stimulation induced expression of pGL3-MBS10, with a more prominent effect observed in membrane-depolarized neurons (Figure 3B and C). Importantly, both KCl and BDNF (Figure 3B and C)-mediated induction were strongly attenuated upon Mef2 knockdown using a previously published Mef2 shRNA construct (Flavell et al, 2006). Simultaneous introduction of an RNAi-resistant Mef2 expression vector (RiRMef2) completely abolished the inhibitory effect of Mef2 knockdown, demonstrating the specificity of the siRNAs. Taken together, results from reporter assays suggest that endogenous Mef2 is required for the depolarization-induced regulation of the miRNA cluster. To confirm that activity-regulated transcription of the endogenous miRNAs is Mef2 dependent, we infected primary neurons using a lentivirus expressing the described Mef2 siRNA. Infected cultures were stimulated with KCl and the levels of selected endogenous miR379–410 pre-miRNAs were assessed by quantitative RT–PCR. For all the miRNAs from the cluster analysed (miR-134 -154, and -376b), Mef2 knockdown significantly reduced the magnitude of KCl-mediated induction (Figure 3D). This effect was specific, as Mef2 knockdown did not affect activity-dependent induction of CREM, a gene that is not regulated by Mef2. Taken together, our data indicate that Mef2 activates miRNA expression in response to neuronal activity by binding to a site located 20 kb upstream of the miRNA cluster. Figure 3.Mef2 is necessary for activity-dependent regulation of the miR379–410 cluster. (A) Binding of Mef2 to MBS10 activates transcription. Reporter genes containing either a wt or mutant MBS10 (MBS10-luc and MBS10-mut-luc, 50 ng) upstream of the luciferase coding region were transfected into cortical neurons (5DIV) along with expression plasmids for constitutively active Mef2 (Mef2-VP16) or a DNA-binding deficient mutant (Mef2-ΔDBD-VP16, 200 ng each). The Mef2 responsive Nur77 reporter was used as a positive control. Luciferase activity was determined and normalized to the internal Renilla control. Fold inductions were derived by dividing the normalized luciferase activity of Mef2-expressing neurons to that of control-transfected neurons. Data represent the mean of three independent experiments+s.d. *P<0.05 (t-test). (B) Mef2 is required for activity-dependent transcription of a MBS10-driven reporter gene. MBS10-luc (100 ng) was transfected into cortical neurons (5DIV) along with a Mef2 shRNA construct (Si-Mef2, 5 ng) and/or an RNAi-resistant Mef2 expression construct (RiRMef2, 100 ng). Neurons were either left untreated or membrane depolarized (57 mM KCl, 6 h). Luciferase activity was determined and normalized to the internal Renilla control. Fold inductions were derived by dividing the normalized activity of KCl-treated neurons to that of untreated neurons. Data represents the mean of three independent experiments+s.d. *P<0.05 (t-test). (C) Mef2 is required for BDNF-dependent transcription of a MBS10-driven reporter gene. Neurons were transfected and treated as in B). Data represents the mean of at least three independent experiments+s.d. *P<0.05 (t-test). (D) Regulation of activity-dependent transcription of the endogenous miRNAs of the miR379–410 cluster by Mef2. Hippocampal neurons were infected with lentiviruses expressing either an shRNA directed against Mef2 (Si-Mef2) or against an unrelated sequence (Si control). Neurons were stimulated with KCl for up to 6 h, total RNA was isolated at the indicated time points and analysed by quantitative RT–PCR using primers specific for miR379–410 precursors, Arc (positive control) and CREM (negative control). For miR-134, one representative out of three independent experiments is shown. Download figure Download PowerPoint Activity-dependent expression of miR379–410 cluster miRNAs is necessary for dendritic development A large number of studies have provided evidence that activity-dependent transcription during early stages of synaptic development has a critical function in dendritic outgrowth (Whitford et al, 2002). To address the physiological relevance of activity-dependent transcription of the miR379–410 cluster, we therefore tested whether perturbation of miR379–410 members affected the ability of neurons to elaborate the dendritic tree in response to activity. To mimic activity-dependent dendritogenesis in vitro, cultured hippocampal neurons (DIV7) were treated for 6 h with KCl or BDNF and analysed by Sholl analysis (see Materials and methods) at DIV10 (Wayman et al, 2006). Both membrane depolarization and BDNF treatment increased the complexity of the dendritic arbour (Figure 4A, left panel) as indicated by a higher number of branches and an increase in the total dendritic length. To obtain a quantitative estimate of activity-dependent changes in complexity, we calculated an induction index by dividing the total number of intersections derived from the Sholl analysis of stimulated neurons to that of unstimulated neurons. Treating neurons with a specific miR-134 anti-miR completely abolished the KCl- and BDNF-mediated increase in dendritic complexity, but had no significant effect on dendrites under basal conditions (Figure 4A–C; Supplementary Figure S1). An antisense oligonucleotide of unrelated sequence (anti-miR control) had no effect on dendritic complexity under both basal and stimulated conditions, demonstrating the specificity of the anti-miR-134. Furthermore, an miR-134 antisense inhibitor of a different chemistry (LNA-modified nucleotides) had a similar effect on the induction index after both KCl and BDNF stimulation (Supplementary Figure S2). We next extended our analysis to other miRNAs from the cluster. Of the four miRNAs considered, we found that anti-miR inhibition of two of them (miR-381 and -329), also blocked activity-dependent dendritogenesis (Figure 4D). Interestingly, inhibition of two other cluster members (miR-495 and -541) had no effect on dendritic complexity under our experimental conditions (Figure 4D). The lack of a dendritic phenotype is unlikely due to inefficient inhibition of these miRNAs, as anti-miRs effectively interfered with three different miRNAs in our sensors assays including miR-541 (Figure 1F). Thus, activity-dependent expression of multiple, but not all miRNAs from the miR379–410 cluster is required for activity-dependent dendritic growth. The requirement of activity-induced expression of the miR379–410 cluster is further supported by our observation that knock down of its upstream activator Mef2 phenocopies miRNA loss of function (Figure 4E). The negative effect of Mef2 siRNA on KCl-dependent dendritogenesis can be rescued by co-transfection of RiRMef2. These data show a previously unknown function of Mef2 in activity-dependent dendritogenesis and identify miRNAs from the miR379–410 cluster as one of the key mediators of this new role of Mef2. Figure 4.Several miRNAs of the miR371–410 cluster are necessary for activity-dependent dendritogenesis. (A) Hippocampal neurons (4DIV) were transfected with GFP together with the indicated anti-miRs (50 nM). At DIV7, neurons were incubated with 16 mM KCl or 40 ng/ml BDNF for 6 h and dendritic complexity was analysed at DIV10 using Sholl analysis (see Materials and methods for details). Scale bar: 20 μm. (B) Quantification of the dendritic complexity of KCl-treated neurons after miR-134 inhibition. Dendritic complexity was calculated by Sholl analysis and the effect of the KCl stimulation on the dendritic tree is expressed as induction index (total number of intersection in stimulated neurons divided by the total number of intersection under basal condition). Here, 10–16 cells for condition were analysed in each experiment. Data represent the mean of three independent experiments+s.d. *P<0.05 (t-test). (C) Quantification of the dendritic complexity of BDNF-treated neurons after miR-134 inhibition. Conditions and data analysis are the same as in (B). Data represent the mean of at least three independent experiments+s.d. *P<0.05 (t-test). (D) Multiple miR379–410 members are necessary for activity-dependent dendritogenesis. Quantification of dendritic complexity of KCl-treated neurons transfected with the indicated anti-miRs (50 nM) was basically as described in (B).