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• W2000600282 abstract "•The miR-7 sponge is sufficient to block silencing activity of three miR-7 precursors•Cortical-specific miR-7 sponge transgenic mice show microcephaly-like brain defects•Expansion and survival of cortical intermediate progenitors require miR-7 function•miR-7 modifies expression levels of genes in the p53 pathway in the embryonic cortex Proper growth of the mammalian cerebral cortex is crucial for normal brain functions and is controlled by precise gene-expression regulation. Here, we show that microRNA-7 (miR-7) is highly expressed in cortical neural progenitors and describe miR-7 sponge transgenic mice in which miR-7-silencing activity is specifically knocked down in the embryonic cortex. Blocking miR-7 function causes microcephaly-like brain defects due to reduced intermediate progenitor (IP) production and apoptosis. Upregulation of miR-7 target genes, including those implicated in the p53 pathway, such as Ak1 and Cdkn1a (p21), is responsible for abnormalities in neural progenitors. Furthermore, ectopic expression of Ak1 or p21 and specific blockade of miR-7 binding sites in target genes using protectors in vivo induce similarly reduced IP production. Using conditional miRNA sponge transgenic approaches, we uncovered an unexpected role for miR-7 in cortical growth through its interactions with genes in the p53 pathway. Proper growth of the mammalian cerebral cortex is crucial for normal brain functions and is controlled by precise gene-expression regulation. Here, we show that microRNA-7 (miR-7) is highly expressed in cortical neural progenitors and describe miR-7 sponge transgenic mice in which miR-7-silencing activity is specifically knocked down in the embryonic cortex. Blocking miR-7 function causes microcephaly-like brain defects due to reduced intermediate progenitor (IP) production and apoptosis. Upregulation of miR-7 target genes, including those implicated in the p53 pathway, such as Ak1 and Cdkn1a (p21), is responsible for abnormalities in neural progenitors. Furthermore, ectopic expression of Ak1 or p21 and specific blockade of miR-7 binding sites in target genes using protectors in vivo induce similarly reduced IP production. Using conditional miRNA sponge transgenic approaches, we uncovered an unexpected role for miR-7 in cortical growth through its interactions with genes in the p53 pathway. Formation of the mammalian cerebral cortex requires precise regulation of neural progenitor proliferation and differentiation to generate the proper number of postmitotic neurons. Radial glial cells (RGCs) in the ventricular zone (VZ) are tightly regulated to maintain their own population while producing intermediate progenitors (IPs) that migrate to the subventricular zone (SVZ), and subsequently mature neurons that form the cortical plate (CP) (Götz and Huttner, 2005Götz M. Huttner W.B. The cell biology of neurogenesis.Nat. Rev. Mol. Cell Biol. 2005; 6: 777-788Crossref PubMed Scopus (1570) Google Scholar, Kriegstein et al., 2006Kriegstein A. Noctor S. Martínez-Cerdeño V. Patterns of neural stem and progenitor cell division may underlie evolutionary cortical expansion.Nat. Rev. Neurosci. 2006; 7: 883-890Crossref PubMed Scopus (556) Google Scholar, Molyneaux et al., 2007Molyneaux B.J. Arlotta P. Menezes J.R. Macklis J.D. Neuronal subtype specification in the cerebral cortex.Nat. Rev. Neurosci. 2007; 8: 427-437Crossref PubMed Scopus (1159) Google Scholar). Proper control of proliferation, survival, and differentiation of neural progenitors is crucial for the formation of normal cortical architecture (Rakic, 2009Rakic P. Evolution of the neocortex: a perspective from developmental biology.Nat. Rev. Neurosci. 2009; 10: 724-735Crossref PubMed Scopus (983) Google Scholar). The expression patterns and levels of genes that govern these processes are closely regulated by molecular mechanisms that are not well understood. It was recently demonstrated that microRNAs (miRNAs) are critical for proper neural progenitor development during corticogenesis (Bian and Sun, 2011Bian S. Sun T. Functions of noncoding RNAs in neural development and neurological diseases.Mol. Neurobiol. 2011; 44: 359-373Crossref PubMed Scopus (130) Google Scholar, De Pietri Tonelli et al., 2008De Pietri Tonelli D. Pulvers J.N. Haffner C. Murchison E.P. Hannon G.J. Huttner W.B. miRNAs are essential for survival and differentiation of newborn neurons but not for expansion of neural progenitors during early neurogenesis in the mouse embryonic neocortex.Development. 2008; 135: 3911-3921Crossref PubMed Scopus (291) Google Scholar, Kawase-Koga et al., 2010Kawase-Koga Y. Low R. Otaegi G. Pollock A. Deng H. Eisenhaber F. Maurer-Stroh S. Sun T. RNAase-III enzyme Dicer maintains signaling pathways for differentiation and survival in mouse cortical neural stem cells.J. Cell Sci. 2010; 123: 586-594Crossref PubMed Scopus (87) Google Scholar, Nowakowski et al., 2011Nowakowski T.J. Mysiak K.S. Pratt T. Price D.J. Functional dicer is necessary for appropriate specification of radial glia during early development of mouse telencephalon.PLoS ONE. 2011; 6: e23013Crossref PubMed Scopus (47) Google Scholar, Shi et al., 2010Shi Y. Zhao X. Hsieh J. Wichterle H. Impey S. Banerjee S. Neveu P. Kosik K.S. MicroRNA regulation of neural stem cells and neurogenesis.J. Neurosci. 2010; 30: 14931-14936Crossref PubMed Scopus (173) Google Scholar). miRNAs are ∼22 nt, endogenous RNAs that guide the RNA-induced silencing complex (RISC) to target mRNAs (Bartel, 2009Bartel D.P. MicroRNAs: target recognition and regulatory functions.Cell. 2009; 136: 215-233Abstract Full Text Full Text PDF PubMed Scopus (16060) Google Scholar). Once targeted, the RISC is able to block translation of or degrade the mRNA transcript (Krol et al., 2010Krol J. Loedige I. Filipowicz W. The widespread regulation of microRNA biogenesis, function and decay.Nat. Rev. Genet. 2010; 11: 597-610Crossref PubMed Scopus (3630) Google Scholar). This posttranscriptional level of regulation is able to fine-tune the expression of target genes and prevent their inappropriate overexpression (Hobert, 2008Hobert O. Gene regulation by transcription factors and microRNAs.Science. 2008; 319: 1785-1786Crossref PubMed Scopus (730) Google Scholar, Otaegi et al., 2011Otaegi G. Pollock A. Hong J. Sun T. MicroRNA miR-9 modifies motor neuron columns by a tuning regulation of FoxP1 levels in developing spinal cords.J. Neurosci. 2011; 31: 809-818Crossref PubMed Scopus (95) Google Scholar). Many miRNAs are members of families or are expressed from multiple loci and thus give rise to mature miRNAs with identical seed sequences, which makes in vivo analyses (e.g., gene-knockout studies) challenging. Promisingly, a miRNA sponge contains complementary binding sequences for the mature miRNAs, titrating them away from their endogenous targets and in turn knocking down a specific mature miRNA or miRNA family (Ebert et al., 2007Ebert M.S. Neilson J.R. Sharp P.A. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.Nat. Methods. 2007; 4: 721-726Crossref PubMed Scopus (1698) Google Scholar, Gentner et al., 2009Gentner B. Schira G. Giustacchini A. Amendola M. Brown B.D. Ponzoni M. Naldini L. Stable knockdown of microRNA in vivo by lentiviral vectors.Nat. Methods. 2009; 6: 63-66Crossref PubMed Scopus (277) Google Scholar). Thus, a miRNA sponge provides an effective way to examine the roles of multilocus miRNAs using a loss-of-function approach. Here, we generated a conditional miR-7 sponge transgenic mouse model in which miR-7 expression is specifically knocked down in the cortex. Blocking miR-7 function transiently affects RGC proliferation, causes severe defects in the progenitor transition from RGCs to IPs, and reduces the survival of progenitors, resulting in reduced neurogenesis and dramatically smaller cortices. Illumina RNA sequencing revealed upregulation of 162 of miR-7’s predicted target genes in the miR-7 sponge cortex, many of which are in the p53 pathway and control cell differentiation and survival. Our results obtained in miR-7 sponge transgenic mice demonstrate that miR-7 plays a crucial role, partly through modifying the p53 pathway, in controlling neural progenitor specification and survival, and determining cortical size. Our initial microarray screen for miRNAs expressed in mouse developing cortices revealed miR-7 expression at embryonic day 12.5 (E12.5) and postnatal day 0 (P0). Mature miR-7 with highly conserved seed sequences was processed from three precursors—miR-7a-1, miR-7a-2, and miR-7b—that were transcribed from separate loci on chromosomes 13, 7, and 17, respectively, in mice (Figure 1A). To verify the expression levels of miR-7 in embryonic cortices, we performed northern blot analyses and detected mature miR-7 in cortices of E12.5 and throughout development using a locked nucleic acid (LNA) probe for miR-7a, which can also detect miR-7b (Figure 1B). Next, we used real-time quantitative RT-PCR (qRT-PCR) to determine which loci were most highly expressed in developing cortices. Although miR-7a-2 and miR-7b showed low levels of expression, miR-7a-1 was the primary source of miR-7, with expression levels more than 25 times higher than miR-7a-2 and close to 12 times higher than miR-7b in the E15.5 cortex (Figure 1C). To further examine the expression pattern of miR-7 in developing cortices, we used the miR-7a LNA probe for in situ hybridization. miR-7a was expressed in the VZ and SVZ in E12.5 cortices and was maintained there through P0 (Figures 1D–1G). miR-7 expression was also detected in the subplate and the CP in E15.5 and P0 cortices (Figures 1F and 1G). These findings suggest that miR-7 may play an important role in neural progenitor development throughout cortical development. To test the role of miR-7 in cortical development using a loss-of-function approach, we designed a bulged miR-7 sponge to simultaneously block the silencing activity of miR-7 transcribed from all three separate loci (Figure 1H). The miR-7 sponge (7-sp) consisted of 24 narrowly spaced, bulged binding sites for miR-7 (Otaegi et al., 2012Otaegi G. Pollock A. Sun T. An optimized sponge for microRNA miR-9 affects spinal motor neuron development in vivo.Front. Neurosci. 2012; 5: 146Crossref PubMed Scopus (47) Google Scholar). Mature miR-7a and miR-7b sequences differ by only a single base, and this was designed to fall within the bulge region of the sponge, so the sponge should affect the function of all three precursors equally by titrating mature miR-7 away from its endogenous targets and leaving them unbound by miR-7 (Figure S1A). We also designed a scrambled sponge construct (Scr-sp) with the same architecture, except that the binding-site sequence is predicted to be untargeted by any miRNA (Figure 1I) (Gentner et al., 2009Gentner B. Schira G. Giustacchini A. Amendola M. Brown B.D. Ponzoni M. Naldini L. Stable knockdown of microRNA in vivo by lentiviral vectors.Nat. Methods. 2009; 6: 63-66Crossref PubMed Scopus (277) Google Scholar). To test the function of these sponges, we designed a luciferase assay, attaching a 3′ UTR containing the miR-7 targeting site to the luciferase gene. All three precursors of miR-7, but not control miRNA miR-17 or a miR-7 construct with a mutated seed sequence, caused a reduction in luciferase activity (Figures 1J and S1B). The miR-7 sponge was then attached to the 3′ UTR of a gene encoding iCre and coexpressed with the three different miR-7 precursors and the luciferase gene with a miR-7 targeting site in its 3′ UTR. Reduced luciferase activity due to any of the three miR-7 precursors was significantly rescued by the miR-7 sponge, but not by a scrambled sponge (Figure 1J). Additionally, a miR-7 sponge with three mutations in the binding seed sequence was unable to rescue reductions caused by miR-7 (Figure S1B). Our results demonstrate that the miR-7 sponge is able to block the function of miR-7 transcribed from any of the three loci. To examine the function of miR-7 in cortical development in vivo, we generated conditional miR-7 sponge transgenic mice. To make the transgene construct, we used the constitutively active CAG promoter to drive expression of a floxed transcriptional stop signal, followed by a coding gene, destabilized GFP (D2eGFP), with the 24 bulged miR-7 sponge sites inserted as its 3′ UTR (Figure 2A). Transgene injection generated two transgenic founder lines (lines 12 and 17), called miR-7 sponge (7-sp) carrier mice, which showed no distinguishable phenotypes. To activate miR-7 sponge activity in the cortex, miR-7 sponge carrier mice were bred with an Emx1-Cre line expressing Cre in the embryonic dorsal cortical region beginning by E10.5 (Gorski et al., 2002Gorski J.A. Talley T. Qiu M. Puelles L. Rubenstein J.L.R. Jones K.R. Cortical excitatory neurons and glia, but not GABAergic neurons, are produced in the Emx1-expressing lineage.J. Neurosci. 2002; 22: 6309-6314Crossref PubMed Google Scholar). Emx1-Cre:miR-7 sponge transgenic mice, called 7-sp mice, showed detectably smaller cortices even at E12.5, with dramatically reduced cortical size at E15.5 and P0 with 100% penetrance (Figure 2B). Lines 12 and 17 showed indistinguishable phenotypes, so the results given for 7-sp mice are for line 17 only. We used the same system to generate a conditional transgenic scrambled sponge mouse. Transgene injection generated five founder lines and, again, mice that carried the transgene alone had no phenotype. Unlike the case with 7-sp mice, however, activation of the scrambled sponge using the Emx1-Cre line, called Scr-sp mice, caused no discernible changes in the cortex in any founder line at all tested stages, so the results given for Scr-sp mice are for line 38 only (Figure 2B). Our results indicate that blocking miR-7 silencing activity in the cortex causes microcephaly-like brain defects. To demonstrate specific activation of the miR-7 sponge transgene, we expressed a nonfluorescent pCAG-iCre plasmid and a sensor construct for miR-7 in the cortices of E13.5 7-sp carrier mice by in utero electroporation. Wild-type littermates received the same electroporation to serve as a control. The sensor plasmid contained the enhanced GFP (eGFP) and monomeric RFP (mRFP) reporter genes, each transcribed from a separate promoter. eGFP has no 3′ UTR, whereas mRFP contains two binding sites for miR-7 in its 3′ UTR (Figure 2C). Therefore, in electroporated wild-type cortices, all cells that receive the plasmid should express eGFP, whereas mRFP, which is sensitive to miR-7 silencing, will be expressed only in cells where it cannot sense miR-7. In electroporated 7-sp carrier cortices, in cells coelectroporated with iCre, miR-7 should be blocked by the miR-7 sponge transgene, allowing mRFP expression. Meanwhile, a subset of cells may not receive iCre coelectroporation. These cells will still have functioning miR-7 and thus express no mRFP (Figure 2C). At 48 hr after electroporation, only ∼10% of electroporated cells were found to express mRFP in the VZ/SVZ of the control cortex, confirming that most neural progenitors expressed endogenous miR-7, although, interestingly, a subpopulation of cells in the VZ/SVZ did not. On the other hand, in 7-sp carrier cortices, over 30% of electroporated cells expressed mRFP (Figures 2D and 2E). This increase indicates that in cells that normally express miR-7, expression of the sponge is able to block miR-7 function in vivo. As predicted, a population of cells appeared green, likely due to unsuccessful coelectroporation of the iCre plasmid. Conversely, electroporation of a sensor for control miRNA miR-9 revealed strong miR-9 activity in nearly all electroporated cells, with no obvious change when the miR-7 sponge transgene was activated (Figure S2). Our results indicate that once it is activated, the miR-7 sponge transgene is able to specifically block the activity of endogenous miR-7, but not miR-9, in cells in the VZ/SVZ of developing cortices. To examine what may cause reduced cortical size in miR-7 sponge mice, we first assessed whether neural production or cortical layer organization was affected. We examined the expression of Tbr1 (layer 6), Ctip2 (layer 5), and Cux1 (layer 2/3) in P0 7-sp and control cortices (Molyneaux et al., 2007Molyneaux B.J. Arlotta P. Menezes J.R. Macklis J.D. Neuronal subtype specification in the cerebral cortex.Nat. Rev. Neurosci. 2007; 8: 427-437Crossref PubMed Scopus (1159) Google Scholar). The relative positioning of layer markers in the CP was similar to that in the wild-type, suggesting that overall cortical layer organization was not greatly affected by miR-7 blockade (Figure 3). However, each layer examined was thinner, with significantly fewer mature NeuN+ neurons, and there were significant reductions in the number of early-born Tbr1+ and Ctip2+ neurons, and late-born Cux1+ neurons (Figure 3). On the other hand, scrambled sponge activation caused no detectable defects compared with controls using any of the assessed markers, suggesting that ectopic expression of a sponge transcript without miRNA binding sites has no effect on cortical neurogenesis (Figure S3). Moreover, TUNEL staining showed no increase in dying cells in P0 7-sp cortices, suggesting that cell survival is not affected by the miR-7 sponge at postnatal stages. Our results indicate that miR-7 is required for proper neurogenesis, but not overall organization, of both early- and late-born neuronal subtypes in different cortical layers. To understand the causes of reduced neurogenesis in the miR-7 sponge cortex, we examined neural progenitor development. Emx1-Cre is activated by E10.5, so we should be able to detect changes in the number of neural progenitors at E12.5. The number of actively cycling progenitors, measured by Ki67 expression, was slightly reduced, and so was bromodeoxyuridine (BrdU) incorporation in the E12.5 7-sp cortex compared with controls (Figures 4A–4C). The cell-cycle labeling index (LI), however, was similar to that of controls (Figure 4D). The number of PH3+ cells in the M phase of the cell cycle was also not changed (Figures 4E and 4F). Correspondingly, we detected a slight reduction in the number of Pax6+ RGCs; however, the number of cycling RGCs that took up BrdU was similar to that observed in controls, suggesting that the mild defect in RGC numbers was not related to their ability to proliferate (Figures 4G and 4H). By later stages, the progenitor pool recovered from its mild defects. The overall numbers of Ki67+ progenitors and Pax6+ RGCs were restored to control levels in E15.5 7-sp cortices, and cell-cycle parameters remained similar to those in controls (Figures 4I–4P). The numbers and cell-cycle LI of neural progenitors in Scr-sp cortices were similar to those in controls at both E12.5 and E15.5 (Figure S4). Our results indicate that blocking the activity of miR-7 has only mild, transient effects on RGCs in the embryonic cortex. Since the number of RGCs was largely unaffected but neurogenesis was significantly reduced in embryonic miR-7-sp cortices, we assessed whether loss of miR-7 function affects IPs by examining the expression of the IP marker Tbr2. Unlike the case with RGCs, the number of Tbr2+ cells remained significantly reduced in 7-sp cortices from E12.5 through E15.5 (Figures 5A–5K). However, in Scr-sp cortices, the number of Tbr2-expressing IPs was not affected (Figure S5). In 7-sp cortices, the number of cells that coexpressed Pax6 and Tbr2, labeling IPs under transition from RGCs, was halved at both E12.5 and E15.5, indicating that miR-7 function is required for normal IP transition (Figures 5C and 5F). At both E12.5 and E15.5, the number of Tbr2+/BrdU+ cells was also reduced in company with overall Tbr2+ cell reductions, leading to an IP cell-cycle LI that was not significantly different from controls (Figures 5G–5L). These results suggest that although the number of IPs was significantly reduced, the behavior of the existing IPs remained similar to that observed for controls. We next tested whether the survival of neural progenitors was affected by miR-7 blockade using a combination of a TUNEL assay with immunofluorescence for Pax6 or Tbr2. Whereas control or scrambled sponge brains showed little cell death, E12.5 miR-7 sponge brains displayed large numbers of TUNEL+ cells that were mostly localized in the SVZ (Figures 5M, 5N, and S5C). At E15.5, individual TUNEL+ cells could still be seen in miR-7 sponge, but not significantly in Scr-sp cortices (Figures 5O, 5P, and S5F). Most TUNEL+ cells were detected in the SVZ and intermediate zone (IZ), but not in the CP (Figures 5O and 5P). These results suggest that miR-7 function is required for neural progenitors, largely in the SVZ, to differentiate and survive. We next assessed the underlying mechanism of miR-7 regulation in cortical development by identifying its target genes. miRNAs can have many simultaneous targets in vivo, so in order to determine which targets may be responsible for the phenotypes in miR-7 sponge mice, we used an Illumina RNA sequencing approach (Figure 6A). This approach provides a global view of alterations to the transcriptome due to manipulation of miR-7, with the expectation that miR-7 target genes released from regulation by miR-7 sponge will be upregulated. Total RNA was isolated from E12.5 dorsal cortices of three 7-sp and three wild-type littermate embryos, sequenced, and analyzed using GobyWeb (Dorff et al., 2013Dorff K.C. Chambwe N. Zeno Z. Simi M. Shaknovich R. Campagne F. GobyWeb: simplified management and analysis of gene expression and DNA methylation sequencing data.PLoS ONE. 2013; 8: e69666Crossref PubMed Scopus (13) Google Scholar). Overall gene expression was not greatly different from that in the wild-type, with only a few genes exhibiting large deviations from controls (Figure 6B). Upon a detailed analysis of the expressed genes, we found that 419 genes had been consistently upregulated by at least 25%. We performed a Gene Ontology (GO) analysis using GOrilla (Eden et al., 2007Eden E. Lipson D. Yogev S. Yakhini Z. Discovering motifs in ranked lists of DNA sequences.PLoS Comput. Biol. 2007; 3: e39Crossref PubMed Scopus (473) Google Scholar, Eden et al., 2009Eden E. Navon R. Steinfeld I. Lipson D. Yakhini Z. GOrilla: a tool for discovery and visualization of enriched GO terms in ranked gene lists.BMC Bioinformatics. 2009; 10: 48Crossref PubMed Scopus (2244) Google Scholar). Genes related to proliferation, cell death or survival, and differentiation were found to be significantly overrepresented among the upregulated genes, suggesting a molecular basis for the observed neurogenesis defects in 7-sp cortices (Figure 6C). To determine which genes may be direct targets of miR-7, we compared the list of upregulated genes with lists of predicted miRNA targets using five target prediction algorithms (miRWalk, Targetscan, Miranda, miRDB, and RNA22) via the miRWalk tool. A total of 162 out of the 419 upregulated genes were predicted targets of miR-7. We next generated an expected number of genes that a nonspecific miRNA would target in our set of 419 upregulated genes by comparing the 419 genes with target lists for 35 other miRNAs, including the 20 highest-expressed neural miRNAs and additional known neural miRNAs (Chi et al., 2009Chi S.W. Zang J.B. Mele A. Darnell R.B. Argonaute HITS-CLIP decodes microRNA-mRNA interaction maps.Nature. 2009; 460: 479-486Crossref PubMed Scopus (1424) Google Scholar). We found that the mean expected number of predicted targets by these baseline miRNAs is approximately 111 ± 4.2 SEM, which is similar to the number of predicted targets for a heart-specific miRNA, miR-1. However, 162 predicted targets of miR-7 were >2 SDs above the nonspecific expectation (Figure 6D). Together with the findings from our miR-7 sensor assay (Figure 2), these results strongly suggest that the phenotype in miR-7 sponge cortices is due to upregulation of miR-7 target genes and a specific blockade of miR-7 function. We hypothesized that rather than targeting individual genes, miR-7 may attempt to regulate whole pathways by silencing multiple genes within that pathway. To test this, we performed a KEGG pathway analysis on the 162 genes that were both upregulated and predicted miR-7 targets using David 6.7 (Figures 6E and S6; Huang et al., 2009Huang W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4: 44-57Crossref PubMed Scopus (25851) Google Scholar). This analysis uncovered that six out of 162 genes are annotated as being part of the p53 signaling pathway, a highly significant overrepresentation. Agilent Literature Search Software v2.8 and a subsequent manual search on PubMed confirmed these six genes and more, showing that a total of 19 out of 162 miR-7 target genes fall into the p53 pathway (Figure 6F). According to the GO annotations of these genes, most were involved in regulating cell-cycle arrest, cell death, and cell differentiation, suggesting underlying causes of miR-7-sp cortical defects. To confirm that these predicted genes can be directly targeted by miR-7, we used luciferase assays, attaching the 3′ UTR of each gene to a luciferase reporter. We selected five predicted miR-7 target genes that are associated with the p53 pathway and have known functions in regulating differentiation or survival in neural development: a cytosolic adenylate kinase Ak1, the apoptotic activator Pmaip1 (also known as Noxa), the CDK inhibitor Cdkn1a (also known as p21), transcription factor Klf4, and the cyclin Ccng1 (Akhtar et al., 2006Akhtar R.S. Geng Y. Klocke B.J. Latham C.B. Villunger A. Michalak E.M. Strasser A. Carroll S.L. Roth K.A. BH3-only proapoptotic Bcl-2 family members Noxa and Puma mediate neural precursor cell death.J. Neurosci. 2006; 26: 7257-7264Crossref PubMed Scopus (55) Google Scholar, van Lookeren Campagne and Gill, 1998van Lookeren Campagne M. Gill R. Tumor-suppressor p53 is expressed in proliferating and newly formed neurons of the embryonic and postnatal rat brain: comparison with expression of the cell cycle regulators p21Waf1/Cip1, p27Kip1, p57Kip2, p16Ink4a, cyclin G1, and the proto-oncogene Bax.J. Comp. Neurol. 1998; 397: 181-198Crossref PubMed Scopus (119) Google Scholar, Noma et al., 1999Noma T. Yoon Y.-S. Nakazawa A. Overexpression of NeuroD in PC12 cells alters morphology and enhances expression of the adenylate kinase isozyme 1 gene.Brain Res. Mol. Brain Res. 1999; 67: 53-63Crossref PubMed Scopus (20) Google Scholar, Qin and Zhang, 2012Qin S. Zhang C.-L. Role of Kruppel-like factor 4 in neurogenesis and radial neuronal migration in the developing cerebral cortex.Mol. Cell. Biol. 2012; 32: 4297-4305Crossref PubMed Scopus (46) Google Scholar). Furthermore, all of these genes showed significant upregulation of expression levels in vivo due to loss of miR-7 function (Figure S6). When coexpressed with miR-7 in vitro, the 3′ UTRs of each of these genes were targeted by miR-7a, resulting in a significant reduction of luciferase activity (Figure 7A ). Luciferase reductions were not induced by coexpression with a control miRNA miR-17 (with the exception of p21, a known target of miR-17) or by a mutated miR-7 (Figure 7A; Wong et al., 2010Wong P. Iwasaki M. Somervaille T.C.P. Ficara F. Carico C. Arnold C. Chen C.-Z. Cleary M.L. The miR-17-92 microRNA polycistron regulates MLL leukemia stem cell potential by modulating p21 expression.Cancer Res. 2010; 70: 3833-3842Crossref PubMed Scopus (156) Google Scholar). Our results indicate that these five genes in the p53 pathway are putative targets for miR-7. We next assessed whether upregulation of these genes in vivo contributes to some of the phenotypes found in miR-7-sp cortices. The full-length cDNA for Ak1 or p21 was ectopically expressed in E13.5 cortices by in utero electroporation, and embryos were analyzed after 24 hr. Consistent with the 7-sp cortical phenotype, ectopic expression of either Ak1 or p21 had no effect on the percentage of electroporated cells expressing Pax6 relative to electroporation with an empty pCAGIG vector. However, there was a significant reduction in the relative percentage of Tbr2-expressing cells, suggesting a specific reduction in IP generation (Figures 7B and 7C). Activated Caspase3 expression was not significantly altered due to ectopic expression of Ak1 or p21. These results suggest that suppression of Ak1 and p21 is necessary for successful generation of IP. Finally, to examine the specific interaction between miR-7 and target genes in vivo, we coelectroporated pCAGIG with LNA target protectors—oligos designed to bind specifically to the miR-7 binding site in the 3′ UTR of Ak1 or p21, preventing miR-7 from silencing its targets. Additionally, we generated a control protector against an alternate site on the Ak1 3′ UTR, which should not affect miR-7 binding. Electroporation of the control protector elicited no changes relative to the no-oligo condition. Electroporation of protectors that blocked miR-7’s interaction with Ak1 or p21, however, closely mimicked overexpression of these genes, with no changes in the relative percentage of Pax6+ cells, and slight but significant reductions in the percentage of Tbr2+ cells (Figures 7D and 7E). Again, activated Caspase3 expression was not significantly altered. Altogether, these results demonstrate that miR-7 function is crucial for neural progenitors to successfully produce IPs, and that this process is partly mediated by the p53 pathway genes Ak1 and p21 in the developing cortex. Because proper cortical size is essential for brain functions, identifying molecules that control cortical growth will help us understand how brain malformations occur. We have generated a mouse model in which the activity of miR-7, processed from all three primary loci, is specifically knocked down i" @default.
• W2000600282 created "2016-06-24" @default.
• W2000600282 creator A5010045617 @default.
• W2000600282 creator A5020250217 @default.
• W2000600282 creator A5040217973 @default.
• W2000600282 creator A5060215226 @default.
• W2000600282 creator A5073719861 @default.
• W2000600282 date "2014-05-01" @default.
• W2000600282 modified "2023-09-26" @default.
• W2000600282 title "Growth of the Developing Cerebral Cortex Is Controlled by MicroRNA-7 through the p53 Pathway" @default.
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