Micro RNA ‐34/449 controls mitotic spindle orientation during mammalian cortex development
2016; Springer Nature; Volume: 35; Issue: 22 Linguagem: Inglês
10.15252/embj.201694056
ISSN1460-2075
AutoresJuan Pablo Fededa, Christopher Esk, Beata E. Mierzwa, Rugilė Stanytė, Shuiqiao Yuan, Huili Zheng, Klaus Ebnet, Wei Yan, Juergen A. Knoblich, Daniel W. Gerlich,
Tópico(s)Genomics and Chromatin Dynamics
ResumoArticle5 October 2016Open Access Source DataTransparent process MicroRNA-34/449 controls mitotic spindle orientation during mammalian cortex development Juan Pablo Fededa Corresponding Author Juan Pablo Fededa [email protected] Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Christopher Esk Christopher Esk Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Beata Mierzwa Beata Mierzwa Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Rugile Stanyte Rugile Stanyte Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Shuiqiao Yuan Shuiqiao Yuan Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA Search for more papers by this author Huili Zheng Huili Zheng Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA Search for more papers by this author Klaus Ebnet Klaus Ebnet Institute-associated Research Group "Cell Adhesion and Cell Polarity", Institute of Medical Biochemistry, ZMBE, Münster, Germany Search for more papers by this author Wei Yan Wei Yan Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA Search for more papers by this author Juergen A Knoblich Juergen A Knoblich Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Daniel W Gerlich Corresponding Author Daniel W Gerlich [email protected] orcid.org/0000-0003-1637-3365 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Juan Pablo Fededa Corresponding Author Juan Pablo Fededa [email protected] Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Christopher Esk Christopher Esk Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Beata Mierzwa Beata Mierzwa Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Rugile Stanyte Rugile Stanyte Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Shuiqiao Yuan Shuiqiao Yuan Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA Search for more papers by this author Huili Zheng Huili Zheng Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA Search for more papers by this author Klaus Ebnet Klaus Ebnet Institute-associated Research Group "Cell Adhesion and Cell Polarity", Institute of Medical Biochemistry, ZMBE, Münster, Germany Search for more papers by this author Wei Yan Wei Yan Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA Search for more papers by this author Juergen A Knoblich Juergen A Knoblich Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Daniel W Gerlich Corresponding Author Daniel W Gerlich [email protected] orcid.org/0000-0003-1637-3365 Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria Search for more papers by this author Author Information Juan Pablo Fededa *,1, Christopher Esk1, Beata Mierzwa1, Rugile Stanyte1, Shuiqiao Yuan2, Huili Zheng2, Klaus Ebnet3, Wei Yan2, Juergen A Knoblich1 and Daniel W Gerlich *,1 1Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), Vienna, Austria 2Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, NV, USA 3Institute-associated Research Group "Cell Adhesion and Cell Polarity", Institute of Medical Biochemistry, ZMBE, Münster, Germany *Corresponding author. Tel: +43 1 7904 44762; E-mail: [email protected] *Corresponding author. Tel: +43 1 7904 44760; E-mail: [email protected] The EMBO Journal (2016)35:2386-2398https://doi.org/10.15252/embj.201694056 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Correct orientation of the mitotic spindle determines the plane of cellular cleavage and is crucial for organ development. In the developing cerebral cortex, spindle orientation defects result in severe neurodevelopmental disorders, but the precise mechanisms that control this important event are not fully understood. Here, we use a combination of high-content screening and mouse genetics to identify the miR-34/449 family as key regulators of mitotic spindle orientation in the developing cerebral cortex. By screening through all cortically expressed miRNAs in HeLa cells, we show that several members of the miR-34/449 family control mitotic duration and spindle rotation. Analysis of miR-34/449 knockout (KO) mouse embryos demonstrates significant spindle misorientation phenotypes in cortical progenitors, resulting in an excess of radial glia cells at the expense of intermediate progenitors and a significant delay in neurogenesis. We identify the junction adhesion molecule-A (JAM-A) as a key target for miR-34/449 in the developing cortex that might be responsible for those defects. Our data indicate that miRNA-dependent regulation of mitotic spindle orientation is crucial for cell fate specification during mammalian neurogenesis. Synopsis A high-throughput imaging screen identifies a set of miRNAs that control mitotic spindle orientation in neuronal progenitors and radial glial cells and whose expression is required for normal cortical development in mouse. An in vitro screen identified miR-34/449 as a candidate cortical miRNA involved in mitotic spindle orientation. miR-34/449 is required for normal neurogenesis during mouse cortical development. The production of neurogenic intermediate progenitors depends on miR-34/449, which controls mitotic spindle orientation in radial glial cells. miR-34/449 regulates spindle orientation at least in part by directly targeting and inhibiting JAM-A. Introduction MicroRNAs (miRNAs) are small noncoding RNAs, which regulate gene expression posttranscriptionally by influencing mRNA stability and/or translation (Ambros, 2004; Du & Zamore, 2005; Kim, 2005). miRNAs are required for brain development in mammals, and growing evidence suggests that they play key roles during cortical neurogenesis (Bian & Sun, 2011). General inhibition of miRNA biogenesis through the conditional deletion of Dicer during early mouse development suppressed the generation of cortical neurons, but not the maintenance of the neural progenitor pool (De Pietri Tonelli et al, 2008; Kawase-Koga et al, 2009; Nowakowski et al, 2011). Since cortical neurons are generated from radial glial cells, these findings suggest that radial glial cell specification might be under the control of miRNAs. Two miRNA families, miR-17~92 and miR-7, were shown to promote the self-renewal of radial glial cells (Bian et al, 2013; Nowakowski et al, 2013; Fei et al, 2014) and the survival and differentiation of neural progenitors (Pollock et al, 2014), respectively. The miR-34b/c and miR-449 cluster encodes for six miRNAs, which are also relevant for brain development, as their deletion in mice reduced brain size and caused defects in basal forebrain structures (Wu et al, 2014). Yet, how these miRNAs contribute to specific stages of cortical development has remained unclear. Mammalian corticogenesis relies on a precise regulation between the generation of neural progenitors and their differentiation (Gotz & Huttner, 2005). This balance is necessary to produce the large number of neurons that shape the multilayered cortical structure. At the onset of cortical neurogenesis, the expansion of neural progenitors by symmetric cell divisions declines and cortical neurons are generated through a series of asymmetric mitoses (Gotz & Huttner, 2005; Lancaster & Knoblich, 2012; Williams & Fuchs, 2013). After their amplification, neuroepithelial progenitors convert into radial glial progenitors, which migrate their nuclei to divide at the apical surface of the ventricular zone (Taverna & Huttner, 2010). Radial glial progenitors then generate cortical neurons by two different modes of asymmetric cell division (Malatesta et al, 2000; Anthony et al, 2004). Direct neurogenesis generates one radial glial progenitor and one neuron, whereas indirect neurogenesis generates one radial glial progenitor and an intermediate progenitor cell (also called basal progenitor), which ultimately generates a pair of neurons (Noctor et al, 2001, 2004; Calegari et al, 2002; Haubensak et al, 2004; Miyata et al, 2004). The degree of asymmetry in cell divisions of radial glial progenitors is influenced by mitotic spindle orientation (Lancaster & Knoblich, 2012; Williams & Fuchs, 2013). During early corticogenesis, parallel orientation of the spindle relative to the ventricular plate is essential for maintaining symmetric divisions. During the subsequent neurogenic phase, oblique and vertical spindle orientation shifts the balance toward indirect neurogenesis (Wynshaw-Boris, 2013; Xie et al, 2013). Several molecular components and their mechanisms of regulating spindle orientation during cortical neurogenesis have been established through prior work (Postiglione et al, 2011; Xie et al, 2013). However, little is known regarding the contribution of miRNAs and posttranscriptional gene regulation to proper spindle orientation. To elucidate the function of specific miRNAs during neural progenitor cell division in cortical development, we screened for candidate regulators of mitosis and subsequently characterized their function in mutant mice. We identified the miR-34/449 family as a critical factor for correct neural progenitor spindle orientation. MiR-34/449 KO mouse embryos had radial glial cells with misoriented spindles, resulting in reduced generation of intermediate progenitors via indirect neurogenesis and smaller cortices. We discovered that miR-449 targets the spindle regulator JAM-A for posttranscriptional repression in vivo. Thus, our findings show that the miR-34/449 family controls spindle orientation and the division of neural progenitors during development, with profound implications in mammalian cortical neurogenesis. Results A screen to identify candidate miRNAs involved in cortical cell division To identify microRNAs (miRNAs) regulating spindle orientation during cortical neurogenesis, we developed an in vitro screening approach to search for candidates that affect cell division. We assayed mitotic duration in a HeLa cell line stably expressing a chromatin marker (histone 2B fused to a red fluorescent protein; H2B–mCherry) and a nuclear import substrate (importin-β-binding domain of importin-α fused to monomeric enhanced green fluorescent protein; IBB–eGFP) using live-cell microscopy (Schmitz et al, 2010). We individually transfected cells with a library of 135 miRNA-mimicking oligomers (miRNA mimics) representing all miRNAs expressed during mammalian corticogenesis (Yao et al, 2012) and, after 48-h incubation, recorded 24-h time-lapse movies for each miRNA mimic (Fig 1A). Mitotic duration was automatically determined for each dividing cell using supervised machine learning (Held et al, 2010). A miR-449b mimic caused the most substantial mitotic delay in this screen. MiR-449a and miR-34c mimics, which belong to the same miRNA family (Kozomara & Griffiths-Jones, 2014), also ranked within the top candidate hits with mitotic delays (Fig 1B and C, Appendix Table S1). Figure 1. Screen for candidate miRNAs involved in cell division Live-cell imaging assay to detect mitotic perturbations. HeLa cells stably expressing a chromatin marker (H2B–mCherry; red) and a nuclear import substrate (IBB–eGFP; green) were imaged by automated live-cell microscopy and the duration of mitosis from prometaphase to anaphase onset was automatically determined for each dividing cell based on the time from nuclear envelope breakdown (mitotic entry) until nuclear reassembly (mitotic exit) (Schmitz et al, 2010). Scale bar, 10 μm. miRNA mimic screen for miRNAs that regulate mitosis genes. miRNA mimics from a library of all embryonic cortically expressed miRNAs were transfected individually into HeLa cells and prometaphase to anaphase onset duration was determined as in (A). Individual points correspond to the mean z-score of the duration of prometaphase to anaphase onset determined in 2 independent experimental replicates for a given miRNA mimic. Cumulative histograms of mitotic progression for control cells and for cells transfected with miRNA mimics. Nuclear envelope breakdown is at t = 0 min (n ≥ 71 in all conditions). Confocal time-lapse microscopy images of HeLa cells stably expressing H2B–mCherry (red) and a microtubule marker (α-Tub–eGFP; green) 48 h after transfection of miR-449a mimic, or nontargeting control siRNA. Scale bar, 10 μm. Quantification of spindle rotation and duration from prometaphase to anaphase onset in time-lapse movies as in (D). miR-449a mimic promotes spindle rotation. Duration from prometaphase to anaphase onset was defined as the time from nuclear envelope breakdown to anaphase onset. Spindle rotation during metaphase was measured as described in Materials and Methods. Individual data points correspond to single cells (n ≥ 72 in all conditions). Normality was tested with Kolmogorov–Smirnov test. Variance between samples was tested using F-test. Significance was tested by Welch's t-test: P-value = 2.598e-06 comparing spindle rotation of negative control vs. miR-449a mimic-transfected cells (all data points are compared). P-value = 0.0005621 for cells with prometaphase–anaphase onset duration lower than 60 min. Source data are available online for this figure. Source Data for Figure 1 [embj201694056-sup-0003-SDataFig1.xlsx] Download figure Download PowerPoint To further characterize the cellular defects associated with the mitotic delays, we investigated spindle rotation dynamics by confocal time-lapse microscopy using a HeLa cell line expressing H2B–mCherry and α-tubulin fused to monomeric enhanced green fluorescent protein (meGFP–α-tubulin; Steigemann et al, 2009). Transfection of the miR449a mimic caused excessive spindle rotation in cells that were delayed in mitosis, but also in cells that progressed to anaphase with normal timing (Fig 1D and E). This suggests that excessive mitotic spindle rotation is a direct consequence of miR449a mimic transfection and not necessarily linked to prolonged mitosis. Thus, miRNA-34/449 family members might be involved in mitotic spindle orientation during brain cortex development. miR-34/449 family is expressed in the radial glial cell niche during cortical neurogenesis The six members of the miRNA-34/449 family are expressed from three different loci (Fig EV1A). To investigate the role of miR-34/449 family during cortical development, we first determined whether the different members of the family are expressed in mice during the onset of cortical neurogenesis, at embryonic day 14 (E14). Laser capture microdissection of flash-frozen embryonic brain slices was performed to isolate the ventricular zone of neocortices, where neural progenitors reside (Fig EV1B). The concentrations of each member of the miR-34/449 family were determined by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) and normalized to the concentration of miR-7-a-1, an abundant miRNA that regulates the p53 pathway in neural progenitors (Pollock et al, 2014). We found at least three members of the miR-34/449 family, miR449a, miR34a, and miR34b, expressed at levels similar to those of miR-7-a-1 (Fig 2A). In situ hybridization of E14 cortical slices further showed that miR-34b and miR449a are predominantly expressed in the ventricular and subventricular zone of the neocortex, where neural progenitors reside (Fig 2B and C). Thus, the abundance and expression pattern of miR-34 and miR-449 is consistent with a potential function in neural progenitors. Click here to expand this figure. Figure EV1. miR-34/449 family locus structure and laser capture microdissection procedure Sequence alignments of mature mouse miR-34/449 miRNAs. Blue letters indicate seed sequences. Laser capture microdissection (LCM) of the ventricular zone (VZ) of mouse cortex at embryonic day E14. Representative images of pre- and post-microdissection. Scale bar, 300 μm. Download figure Download PowerPoint Figure 2. miR-34/449 family is expressed in neural progenitors and is required for normal cortex development A. The expression levels of endogenous miR-34/449 family members were measured by RT–qPCR in ventricular zone samples derived by laser microdissection of mouse cortices at E14. The levels of the different miR-34/449 family members and miR-7a-1, a highly expressed miRNA relevant in cortical progenitor biology, were determined. All concentrations were normalized (norm.) using miR-7a-1 concentration (n = 8 cortices, 2 different litters). Error bars indicate standard error. B, C. Expression analysis of miR-34/449 by in situ hybridization using locked nucleic acid (LNA) probes in wild-type cortices at E14. Mature miR-449, miR-34b, and miR-34c are preferentially expressed in the subventricular (SVZ) and ventricular (VZ) zones of the neocortex. Scale bar, 50 μm (B), 10 μm (C). D, E. Brains of adult mice (P23) and quantification of brain weight. Dots indicate individual brains; red line indicates median. Mice lacking miR-449abc and miR-34bc (DKO) or miR-449abc, miR-34bc, and miR-34a (TKO) have significantly smaller brains compared to littermate controls (Het). Significance was tested by pairwise t-test with Bonferroni correction; Het (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters), **P-value = 0.00215; Het (n = 6 brains, 2 different litters) vs. TKO (n = 4 brains, 2 different litters), ***P-value = 0.00054. F, G. Confocal images of coronal brain sections (P23) and quantification of cortex width from miR-34/449 DKO and TKO mice and littermate controls (Het). Sections were stained with DAPI. DKO or TKO mice have significantly thinner cortices compared to littermate controls (Het) in adult mice (P23). Significance was tested by pairwise t-test with Bonferroni correction; Het (n = 6 brains, 2 different litters) vs. DKO (n = 4 brains, 2 different litters), ***P-value = 0.00084; Het (n = 6 brains, 2 different liters) vs. TKO (n = 4 brains, 2 different litters), **P-value = 0.00211. Scale bar, 500 μm. Source data are available online for this figure. Source Data for Figure 2 [embj201694056-sup-0004-SDataFig2.xlsx] Download figure Download PowerPoint Deletion of miR-34/449 perturbs cortical development Genetic deletion of the miR-34/449 family in mice was previously shown to cause reduced brain size (Song et al, 2014) and perturbed development of intermediate forebrain structures (Wu et al, 2014), yet the specific developmental defects underlying these phenotypes were not determined. To further dissect the function of miR-34/449 during brain development, we generated double and triple knockout mice (DKO and TKO) of miR-449abc/34bc and miR-449abc/34a/34bc loci, respectively, crossing previously generated miR-449abc and miR-34a/34bc KO mice (Bao et al, 2012; Concepcion et al, 2012). This revealed that the reduced brain size of the miR-34/449 DKO/TKO mice (Fig 2D and E) was largely due to significantly thinner cortices, compared to heterozygote litter controls in young postnatal day 23 (P23) mice (Fig 2F and G). The brain size and cortical thickness phenotypes of miR-34/449 DKO and TKO mice were indistinguishable, indicating that miR-34a does not compensate for the deletion of other miRNA family members. For further loss-of-function phenotype characterization, we therefore combined DKO/TKO littermates into one group (subsequently referred to as KO). Together, these data show that miR-34/449 has an important function in the development of the mouse brain cortex. miR-34/449 regulates neurogenesis in the mouse cortex Changes in cortical thickness observed at P23 in miR-34/449 KO mice might result from defective cortical neurogenesis during embryonic development. To test this, we first imaged brain sections of mouse embryos at embryonic day 16 (E16) stained for neuronal markers of the upper cortical layers, Satb2 (layers II–III) and Ctip2 (layer V). The number of Satb2+ cells, which are still migrating basally at this stage (Britanova et al, 2008) (Figs 3A and B, and EV3A), and Ctip2+ cells (Figs 3C and D, and EV3A) was significantly reduced in miR-34/449 KO (i.e., grouped DKO and TKO) mice, indicating a delay in late neuron generation during cortical development. We next imaged brain sections at E14 stained with the deep layer neuronal marker Tbr1 (layer VI). This showed that the formation of the first layer during corticogenesis was also significantly impaired in miR-34/449 mice (Figs 3E and F, and EV3B). Altogether, these data show that miR-34/449 is important for the generation of several cortical layers at different stages during cortical neurogenesis. Figure 3. miR-34/449 family is required for timely cortical neurogenesis A, B. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Satb2-antibody to label neurons of layers II–IV (green) and DAPI to label all cell nuclei (blue). Quantification of the number of Satb2-positive cells per 100 μm ventricular zone surface (n = 4 brains per genotype group, 2 independent litters). *P-value = 0.01146 (Het vs. KO). C, D. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Ctip2 antibody to label V neurons and DAPI to label all cell nuclei (blue). Images were taken from same brain slices as shown in (A). Scale bars: 50 μm. Quantification of the number of Ctip2-positive cells per 100 μm ventricular zone surface (n = 4 brains per genotype group, 2 independent litters). *P-value = 0.03224 (Het vs. KO). E, F. Confocal images of coronal sections from E14 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Tbr1 antibody to label layer IV neurons and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr1-positive cells per 100 μm ventricular zone surface (n = 3 and 4 brains per genotype group from 2 independent litters). *P-value = 0.01392 (Het vs. KO). G, H. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Tbr2 antibody to label intermediate progenitors and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr2-positive cells per 100 μm ventricular zone surface (n = 7 brains for each genotype group, 5 independent litters). *P-value = 0.01647 (Het vs. KO). I, J. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with anti-Pax6 antibody to label radial glia progenitors and DAPI to label all cell nuclei (blue). Quantification of the number of Tbr2-positive cells per 100 μm ventricular zone surface (n = 5 brains per genotype group, 3 independent litters). **P-value = 0.002768 (Het vs. KO). K. 3D reconstruction of a dividing radial glial progenitor at early anaphase. A coronal section of an E14 brain of a heterozygous control mouse was stained with anti-phospho-vimentin antibody (red), anti-γ-tubulin antibody (green/yellow), phalloidin (magenta), and DAPI (blue) and imaged by 3D confocal microscopy. "s" indicates spindle axis, and "α" indicates angle relative to ventricular surface plane, which was determined by a vector path as indicated by thin white lines located on the left side of the image. Yellow dots highlight the centrosomes of the spindle poles from the analyzed dividing cell. L. Quantification of spindle orientation in radial glial cells as in (K) for E14 brains of miR-34/449 KO and littermate controls (Het). Each dot represents a single dividing cell; ***P-value = 3.166e-05 (Het vs. KO) (n = 131 vs. 107 cells, n = 4 brains per genotype group, 2 independent litters). Data information: Scale bars: 50 μm (A, C, E, G, I) and 5 μm (K). Bars indicate mean ± SEM. Data were normalized (norm.) to the ventricular zone surface analyzed (100 μm) and relativized to the heterozygous control average value. Statistical significance was tested by Welch's t-test. Source data are available online for this figure. Source Data for Figure 3 [embj201694056-sup-0005-SDataFig3.xlsx] Download figure Download PowerPoint It was previously shown that the observed reduction in cortical thickness in Dicer-ablated cortices was, in part, the result of the apoptosis of newborn neurons (De Pietri Tonelli et al, 2008). To determine whether the neurogenic defects observed in miRNA-34/449 KO mice were the result of the apoptosis of differentiating neurons, we imaged brain sections of mouse embryos at E16 stained for TUNEL or activated caspase-3 (Fig EV2A and B). Cell death was not detected in both miR-34/449 KO mice and control littermates, suggesting that the deficiency in neuron generation in the miR-34/449 KO is not the secondary effect of cell death/apoptosis during the differentiation process. Click here to expand this figure. Figure EV2. Apoptosis and cell cycle phenotypes in miR-34/449 KO mouse neocortices A, B. Confocal images of coronal sections from E16 brains of miR-34/449 KO mice and littermate controls (Het), stained with TUNEL (green) or activated caspase-3 (red) and DAPI to label all cell nuclei (blue). Cells stained for TUNEL and activated caspase-3 are not detectable in mutant and control brains. Sections were counterstained with DAPI (blue). Scale bar, 50 μm. C. Scheme of BrdU incorporation protocol in female pregnant mice to measure cell cycle exit rates of neural progenitors in the embryonic cortex. D. Confocal images of coronal sections from miR-34/449 KO mice brain compared with littermate controls (Het) in E16 embryos labeled for BrdU (24 h, red) and stained for Ki67 (green). Scale bar, 50 μm. E. Mitotic exit rates in miR-34/449 KO mice brain compared with littermate controls (Het) at E16. Cells exiting cell cycle during the BrdU incorporation (24 h) are BrdU positive but Ki67 negative. BrdU labeling and Ki67 labeling show no difference between KO embryos compared to littermate controls (n = 4 brains for each genotype group, 2 independent litters). Data were normalized (norm.) to the ventricular zone surface analyzed (100 μm) and relativized to the heterozygous control average value. P-value = 0.8294 (Het vs. KO); red bar indicates median. Significance was tested by Welch's t-test. F. Scheme of BrdU and EdU incorporation protocol in female pregnant mice to measure cell cycle duration and S phase duration in neural progenitors in the embryonic cortex. G. Confocal images of coronal sections from miR-34/449 KO mice brain compared with littermate controls (Het) in E15 embryos labeled for EdU (3 h, green) and BrdU (0.5 h, red). Scale bar, 50 μm. H, I. S phase duration and cell cycle duration in miR-34/449 KO mice brain compared with littermate controls (Het) at E15. S phase duration and cell cycle duration were calculated as described in Materials and Methods. BrdU labeling and EdU labeling show no difference between KO embryos compared to littermate controls (n = 3 brains for each genotype group, 2 independent litters); P-value = 0.9324 (S phase duration, Het vs. KO), P-value = 0.9324 (cell cycle duration, Het vs. KO); red bar indicates median. Significance was tested by Welch's t-test. Source data are available online for this figure. Download figure Download PowerPoint In some cell types, miR-34 expression is regulated by the p53 pathway that activates G1 arrest (Chivukula & Mendell, 2008) and thus might control the rates of cell cycle exit during neuronal development. To test whether the lower number of cortical neurons in miR-34/449 KO mice correlate with perturbed cell cycle progression, we injected day 15 pregnant female mice with 5-bromo-2′-deoxyuridine (BrdU) for 24 h, fixed E16 embryo brains, and performed double immunostaining for BrdU and the proliferation marker Ki67 (Fig EV2C and D). The abundance of BrdU+ and Ki67+ cells was indistinguishable in miR-34/449 KO and littermate heterozygous controls (Fig EV2E), suggesting that miR-34/449 is not relevant for cell cycle exit in neural progenitors. To further test whether the neurogenic defects in miR-34/449 mice correlate with perturbed S phase and/or overall cell cycle duration, we consecutively injected
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