Repression of Irs2 by let‐7 mi RNA s is essential for homeostasis of the telencephalic neuroepithelium
2020; Springer Nature; Volume: 39; Issue: 21 Linguagem: Inglês
10.15252/embj.2020105479
ISSN1460-2075
AutoresVirginia Fernández, Maria Ángeles Martínez‐Martínez, Anna Prieto‐Colomina, Adrián Cárdenas, Rafael Soler, Martina Dori, Ugo Tomasello, Yuki Nomura, José P. López‐Atalaya, Federico Calegari, Vı́ctor Borrell,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle28 September 2020Open Access Transparent process Repression of Irs2 by let-7 miRNAs is essential for homeostasis of the telencephalic neuroepithelium Virginia Fernández Virginia Fernández orcid.org/0000-0001-6476-6134 Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Maria Ángeles Martínez-Martínez Maria Ángeles Martínez-Martínez Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Anna Prieto-Colomina Anna Prieto-Colomina Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Adrián Cárdenas Adrián Cárdenas Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Rafael Soler Rafael Soler Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Martina Dori Martina Dori CRTD-Center for Regenerative Therapies, School of Medicine, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Ugo Tomasello Ugo Tomasello Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Yuki Nomura Yuki Nomura Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author José P López-Atalaya José P López-Atalaya Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Federico Calegari Federico Calegari orcid.org/0000-0002-3703-2802 CRTD-Center for Regenerative Therapies, School of Medicine, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Víctor Borrell Corresponding Author Víctor Borrell [email protected] orcid.org/0000-0002-7833-3978 Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Virginia Fernández Virginia Fernández orcid.org/0000-0001-6476-6134 Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Maria Ángeles Martínez-Martínez Maria Ángeles Martínez-Martínez Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Anna Prieto-Colomina Anna Prieto-Colomina Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Adrián Cárdenas Adrián Cárdenas Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Rafael Soler Rafael Soler Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Martina Dori Martina Dori CRTD-Center for Regenerative Therapies, School of Medicine, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Ugo Tomasello Ugo Tomasello Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Yuki Nomura Yuki Nomura Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author José P López-Atalaya José P López-Atalaya Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Federico Calegari Federico Calegari orcid.org/0000-0002-3703-2802 CRTD-Center for Regenerative Therapies, School of Medicine, Technische Universität Dresden, Dresden, Germany Search for more papers by this author Víctor Borrell Corresponding Author Víctor Borrell [email protected] orcid.org/0000-0002-7833-3978 Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain Search for more papers by this author Author Information Virginia Fernández1,3, Maria Ángeles Martínez-Martínez1, Anna Prieto-Colomina1, Adrián Cárdenas1, Rafael Soler1, Martina Dori2, Ugo Tomasello1, Yuki Nomura1, José P López-Atalaya1, Federico Calegari2 and Víctor Borrell *,1 1Instituto de Neurociencias, Consejo Superior de Investigaciones Científicas & Universidad Miguel Hernández, Sant Joan d'Alacant, Spain 2CRTD-Center for Regenerative Therapies, School of Medicine, Technische Universität Dresden, Dresden, Germany 3Present address: Fondazione Istituto Italiano di Tecnologia (IIT), Genoa, Italy *Corresponding author. Tel: +34 965 919245; E-mail: [email protected] The EMBO Journal (2020)39:e105479https://doi.org/10.15252/embj.2020105479 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 Structural integrity and cellular homeostasis of the embryonic stem cell niche are critical for normal tissue development. In the telencephalic neuroepithelium, this is controlled in part by cell adhesion molecules and regulators of progenitor cell lineage, but the specific orchestration of these processes remains unknown. Here, we studied the role of microRNAs in the embryonic telencephalon as key regulators of gene expression. By using the early recombiner Rx-Cre mouse, we identify novel and critical roles of miRNAs in early brain development, demonstrating they are essential to preserve the cellular homeostasis and structural integrity of the telencephalic neuroepithelium. We show that Rx-Cre;DicerF/F mouse embryos have a severe disruption of the telencephalic apical junction belt, followed by invagination of the ventricular surface and formation of hyperproliferative rosettes. Transcriptome analyses and functional experiments in vivo show that these defects result from upregulation of Irs2 upon loss of let-7 miRNAs in an apoptosis-independent manner. Our results reveal an unprecedented relevance of miRNAs in early forebrain development, with potential mechanistic implications in pediatric brain cancer. Synopsis Dicer1-dependent let-7 microRNAs are critical to repress p53 pathway activity and Irs2 expression levels in the early telencephalic neuroepithelium, which otherwise enters into hyperproliferation and loss of adhesion of progenitor cells, leading to the formation of true rosettes reminiscent of pediatric cancers. Loss of miRNAs in the early embryonic telencephalon of Rx-Cre;Dicer1flox mutant mice produces massive transient apoptosis of progenitor cells, loss of adherens junctions and increased proliferation, leading to the formation of true hyperproliferative rosettes. Rx-Dicer mutants have a significant loss of let-7 family microRNAs, and increased p53 signaling and Irs2 expression. Loss of p53 in Rx-Dicer mutants rescues their phenotype in a partially-penetrant manner. Increased Irs2 expression or blockade of let-7 activity in early wild-type embryos, induce rosette formation in the rostral telencephalon, rescued by overexpression of let-7 and downregulation of Irs-2, respectively. Let-7 miRNAs downregulate the amplification of human cortical progenitor cells by repressing Irs2 expression, consistent with a role of this signaling axis in pediatric cancers with true rosettes. Introduction The development of the telencephalon is a highly complex process involving a sequence of key events. Neurogenesis onsets with the emergence of apical radial glia cells (aRGCs), specialized neuroepithelial cells essential in telencephalic development that serve as neural progenitors and guide for the migration of newborn neurons. aRGCs are highly polarized and extend thin processes attached to the ventricular (apical) and pial (basal) surfaces of the developing telencephalon, with the cell bodies forming the ventricular zone (VZ). Their apical process terminates at the ventricular surface in an end foot, which serves to anchor aRGCs to each other via adherens junctions. This maintains the polarity of aRGCs and the cellular homeostasis and structural integrity of the VZ (Gotz & Huttner, 2005; Marthiens et al, 2010). Upon cell division, aRGCs generate either additional aRGCs, neurons, or basal progenitor cells, which delaminate from the ventricular surface and migrate to the basal side of the VZ, forming the SVZ (Miyata et al, 2004; Noctor et al, 2004, 2008). In mouse, most basal progenitors are intermediate progenitor cells (IPCs), producing the majority of excitatory neurons (Haubensak et al, 2004; Kowalczyk et al, 2009; Taverna et al, 2014). The integrity of the VZ and its apical adherens junction belt is essential for the normal development of the telencephalon, including progenitor cell proliferation and neuron migration (Cappello et al, 2006; Rasin et al, 2007; Taverna et al, 2014). Its disruption due to developmental insult or genetic mutation leads to severe malformations of brain development in humans (Barkovich et al, 2012; Fernandez et al, 2016). Molecular mechanisms involved in the apical anchoring of aRGCs have been identified (Gotz & Huttner, 2005; Cappello et al, 2006; Rasin et al, 2007), but mechanisms regulating gene expression to preserve the global integrity and homeostasis of the neuroepithelial niche remain largely unexplored. Gene expression and function during brain development are finely tuned by a number of regulatory mechanisms (Bae et al, 2015; Nord et al, 2015; Yao et al, 2016). Non-coding RNAs, and particularly microRNAs, are major post-transcriptional regulators of gene expression involved in many developmental processes (Aprea & Calegari, 2015). In the embryonic cerebral cortex, many cell cycle-related proteins are targets of miRNAs (Arcila et al, 2014). Previous mouse models of miRNA loss consistently used conditional mutants where miRNAs are depleted only at mid-late corticogenesis. This caused very limited defects on cell proliferation or neurogenesis, leading instead to massive apoptosis of progenitor cells and postmitotic neurons only at late developmental stages (De Pietri Tonelli et al, 2008; Kawase-Koga et al, 2010; McLoughlin et al, 2012; Nigro et al, 2012; Saurat et al, 2013). As a result, miRNA function in early telencephalic development remains largely unknown. Biogenesis of miRNAs requires processing of pre-miRNAs into mature miRNAs, which in turn depends on the action of the RNase enzyme Dicer1 (Bartel, 2018). Complete loss of Dicer1 in full knockout mouse zygotes leads to early developmental defects and embryonic arrest after gastrulation, around embryonic day 7.5 (E7.5) (Bernstein et al, 2003), which evidences the fundamental importance of miRNAs in early embryonic development. Given this very early lethality, understanding the function of Dicer-dependent miRNAs in telencephalic development, which occurs much later, requires the use of conditional knockouts, crossed with a variety of Cre recombinase-expressing mouse lines (Harfe et al, 2005). Emx1-Cre, Nestin-Cre, and hGFAP-Cre mice have been widely used for studies of embryonic development of the cerebral cortex, taking advantage of their early expression (E9.5, E10.5, and E13.5, respectively) (Zimmerman et al, 1994; De Pietri Tonelli et al, 2008; Kawase-Koga et al, 2010; Saurat et al, 2013; Zhang et al, 2015). Elimination of Dicer with these Cre driver lines has produced a significant variety of phenotypes (Kawase-Koga et al, 2009), but surprisingly, those studies failed to identify significant roles for miRNAs in dorsal telencephalic development prior to E13.5, in spite of the high expression levels of miRNAs since E10.5 (Kloosterman et al, 2006; De Pietri Tonelli et al, 2008). This suggests that upon disruption of the gene locus, both Dicer protein and miRNA levels remain largely unchanged within targeted cells for a long time, or until diluted over consecutive cell cycles. Thus, elucidating the role of miRNAs in embryonic telencephalic development may require the removal of Dicer at much earlier stages than in previous studies. Here, we have studied the role of miRNAs in early telencephalic development by using the Rx-Cre driver mouse line (Swindell et al, 2006), which recombines in the primordium of the telencephalon at E7.5, 3 days earlier than in previous models. Rx-Cre;DicerF/F(Rx-Dicer) mutant embryos displayed mild developmental defects in the neocortex related to increased apoptosis, similar to previous reports (De Pietri Tonelli et al, 2008). However, in the rostral telencephalon the early loss of Dicer led to very severe tissue disorganization and the massive formation of highly proliferative rosettes, which grew caudally. Time-course analyses revealed that rosettes formed by invagination of the rostral neuroepithelium, a process concomitant with apoptosis but independent from it, and due to the loss of apical adherens junctions and increased proliferation. These two aspects of the phenotype emerged from decreased levels of let-7 miRNAs and increased expression of targets that promote apoptosis and/or proliferation: p53 signaling and insulin receptor substrate-2 (Irs2), respectively. The formation of rosettes in Rx-Dicer mutants was prevented by the loss of p53, but this was independent from a loss in apoptosis, as overexpression of Irs2 alone in wild-type embryos was sufficient to induce rosette formation without massively increasing apoptosis. The formation of hyperproliferative rosettes upon Irs2 overexpression was rescued by overexpression of let-7. This was phenocopied by the loss of endogenous let-7 alone, which was then rescued by the loss of function of Irs2. The positive effects of Irs2 on telencephalic progenitor proliferation, and negative of let-7, were phenocopied in human cerebral organoids, indicating that this is a highly conserved mechanism. Our results suggest a general relevance of miRNA dysregulation on the emergence of malformations of early brain development and, potentially, other tissues of ectodermal origin. Results Early loss of telencephalic miRNAs in Rx-Cre;DicerF/F embryos To investigate the roles of miRNAs in early telencephalic development, we circumvented the timing limitation of previous Cre driver lines by using Rx-Cre mice, which express Cre under the control of the regulatory sequences of the transcription factor Rax (Rx) (Swindell et al, 2006). Best known for its specific expression in the developing retina, Rx is first expressed in the anterior neural fold (prospective forebrain) of E7.5 mouse embryos, 3 days earlier than Emx1 (Furukawa et al, 1997). Rx-Cre mice crossed with the Rosa26-tdTomato reporter line (Madisen et al, 2010) demonstrated Cre recombination in the emerging telencephalic vesicles as early as E8.5, later becoming distinctively restricted to the telencephalon (Fig EV1A). Within the telencephalon, tdTomato expression level changed as development progressed, gradually increasing from E11.5 to E17.5/postnatal day 1 (P1; Fig EV1B–D). The basal ganglia expressed the highest levels of tdTomato already at E11.5, with the olfactory bulb (OB) and septum reaching similar levels by E12.5 (Fig EV1C and D). In contrast, tdTomato expression at these early stages was very low in the neocortex, indicating a significantly lower level of Cre recombination (Fig EV1C and D, Table EV1). TdTomato levels in the neocortex increased gradually from E12.5 to E17.5, when reaching statistical similarity with the rest of the telencephalon (Fig EV1D). Nevertheless, the levels of tdTomato expression in the neocortex always tended to be lowest in the caudal and highest in the rostral neocortex, the area where expression reached greater similarity with the OB, septum, and basal ganglia. TdTomato in the thalamus was virtually absent at all ages, in agreement with this region not deriving from the lineage of Rx+ territories (Fig EV1D). Taken together, these analyses showed that Cre recombination in Rx-Cre mouse embryos is most prevalent in the rostral and ventral telencephalon at E11.5 and E12.5, while it is significantly lower in the neocortex, particularly in its medial and caudal aspects. Click here to expand this figure. Figure EV1. Early recombination in the dorsal telencephalon of Rx-Cre mice A, B. Bright field (top row) and TdTomato expression (red) under the Rx3 promoter as seen in whole embryos (A) and brain sections (B) at the embryonic ages indicated. Sections in (B) are in coronal (upper panels) and sagittal plane (bottom panels). BG, basal ganglia; di, diencephalon; H, hippocampus; NCx, neocortex; OB, olfactory bulb; ov, optic vesicle; St, striatum; Th, thalamus; tv, telencephalic vesicle. Scale bars, 500 μm (A), 300 μm (B). C, D. Quantification of Tomato fluorescence intensity upon Rx-Cre recombination in the forebrain areas and at the embryonic stages indicated. BG, basal ganglia; NCx, neocortex; OB, olfactory bulb. Dashed line indicates average level of Tomato fluorescence in Basal Ganglia. Comparison across telencephalic areas at each stage is shown in (D). Data in histograms are mean ± SEM, symbols indicate individual values; n = 3–8 replicates per age. ANOVA followed by Tukey's test; ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Table EV1 for the full set of statistical results. Download figure Download PowerPoint Next, we tested the elimination of Dicer-dependent miRNAs using a conditional Dicerflox/flox mouse line (DicerF/F). ISH against Dicer and miR9, one of the miRNAs most highly expressed in the developing vertebrate brain (Radhakrishnan & Alwin Prem Anand, 2016), demonstrated their substantial reduction in the rostral telencephalon of Rx-Cre;DicerF/F mutants at E12.5, and their absence by E17.5 (Fig EV2). At early developmental stages, the loss of Dicer and miR9 was almost complete and much more prominent in the rostral and ventral telencephalon (OB, septum, and basal ganglia) than in the dorsal telencephalon (neocortex), consistent with the pattern of TdTomato expression upon Rx-Cre recombination described above. Dicer and miR9 were still expressed at medial levels of the dorsal telencephalon by E17.5 (Fig EV2), indicating an incomplete recombination of Dicer in this territory. Given the high abundance of miR9 in the normal mouse embryo brain, its major reduction in expression confirmed the loss of functional Dicer in Rx-Cre;DicerF/F embryos. Click here to expand this figure. Figure EV2. Early loss of telencephalic Dicer and miRNAs in Rx-Cre;DicerF/F mutant mouse embryosSagittal sections of control (DicerF/F) and Rx-Dicer (Rx-Cre;DicerF/F) mutant embryos at E12.5 and E17.5 showing the expression of Dicer1 mRNA and miR-9 in the olfactory bulb (OB) and neocortex (NCx). Dashes line is apical surface. Levels of Dicer and miR9 expression were dramatically reduced in the OB and subpallium of mutants. St, striatum; H, hippocampus; Th, thalamus. Scale bar, 100 μm (E12.5, all images at the same scale), 1 mm (E17.5, all images at the same scale). Download figure Download PowerPoint Massive and transient cell death without loss of proliferation in the rostral telencephalic primordium Previous in vivo and in vitro studies demonstrated that Dicer mutants typically exhibit high levels of cell death, including in the developing neocortex, particularly at intermediate to late embryonic stages (Mott et al, 2007; Raver-Shapira et al, 2007; Davis et al, 2008; De Pietri Tonelli et al, 2008). In contrast, our analysis of Casp3 stains in the E11.5 telencephalic primordium of Rx-Cre;DicerF/F mutant embryos (Rx-Dicer mutants, from hereon) revealed the occurrence of dramatically high levels of apoptosis in the rostral and ventral telencephalon, but not in the neocortex (Fig 1A). This was consistent with the previous reports showing that miRNAs are critical to prevent apoptosis in the developing telencephalon, but in this case at much earlier stages, and also consistent with the greater level of Cre recombination in the rostral and ventral embryonic telencephalon of Rx-Cre mice (Figs EV1 and EV2). Marker analyses showed that apoptosis involved mostly Pax6+ aRGCs (67% of Casp+ cells) and only a small minority of Tbr1+ neurons (8.6% of Casp+ cells; Fig 1B and C). A detailed time-course analysis revealed that apoptotic events started suddenly and at high levels at E11.5, with apoptotic cells arranged in columns that spun the entire thickness of the telencephalic primordium (Fig 1D and E). Remarkably, massive cell death lasted only between E11.5 and E12.5, decreasing suddenly again by E13.5 (Fig 1D and E). Figure 1. Massive progenitor cell apoptosis without global loss of proliferation in rostral telencephalon of Rx-Dicer mutants A. Distribution of Casp3+ cells in rostral and ventral domains of an E11.5 Rx-Dicer mutant embryo. NCx, neocortex; OB, olfactory bulb; Spt, septum. B, C. Marker analysis of Casp3+ cells in the rostral telencephalon of E12.5 Rx-Dicer mutant embryos. Most apoptotic cells are Pax6+ RGCs (solid arrowheads) and not Tbr1+ neurons (open arrowheads). N = 3 replicates per marker. D, E. Distribution and abundance of apoptotic cells (Casp3+) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, and dashed line, apical surface. N = 3 replicates per genotype and age. F, G. Distribution and abundance of mitoses (PH3+) and neurons (Tuj1) in the rostral telencephalic primordium of control and Rx-Dicer mutant embryos at the indicated ages. Dotted line indicates basal surface, and dashed line, apical surface. N = 3–4 replicates per genotype and age. No significant differences were found between control and mutant embryos in apical nor basal mitoses. Data information: Histograms represent mean ± SEM; symbols in plots indicate values for individual embryos; chi-square test (C), t-test (E, G); *P < 0.05, **P < 0.01, ***P < 0.001. Scale bars, 200 μm (A), 40 μm (B), 25 μm (D, F). Download figure Download PowerPoint Next, we reasoned that the dramatically high levels of apoptosis among aRGCs would strongly decrease proliferation. However, anti-PH3 stains demonstrated that the density of apical and basal mitoses was not significantly altered in Rx-Dicer mutant embryos between E10.5 and E13.5 (Fig 1F and G). Together, our results indicated that Rx-Dicer mutant embryos are severely affected by massive apoptosis largely in the rostral telencephalon and between E11.5 and E12.5, affecting mostly aRGCs. Remarkably, in spite of this massive cell death, the density of mitotic events remained unaltered in Rx-Dicer mutants, suggesting that the remaining non-apoptotic progenitor cells may proliferate and self-renew at rates higher than normal. Disorganization of the rostro-ventral telencephalon in Rx-Dicer embryos Next, we investigated the long-term consequences of the high apoptotic levels in the rostral telencephalic primordium (prospective OB) of early Rx-Dicer mutant embryos. Sagittal sections through the brains of Rx-Dicer mutants at E17.5 showed that the OB was much smaller than in control embryos (Fig 2A–C). We confirmed the OB identity and reduction in size by ISH stains for Grm1, a marker of mitral cells in E17.5 embryos, which also revealed that the laminar organization of the OB was largely preserved (Fig 2B and C). In addition to a smaller OB, we observed a general and profound disorganization of the entire rostral–ventral region of the telencephalon in Rx-Dicer mutants, including the prefrontal neocortex and the septum (Fig 2D). These alterations also affected the rostral neocortex, but not its parietal region (Fig EV3A and B), consistent with the greater loss of miRNAs at early embryonic stages in the former. The disorganization of the rostral telencephalon in Rx-Dicer mutants involved an overabundance of Ki67+ progenitor cells in the germinal zones (Fig 2D). A closer examination revealed that Ki67+ cells were in fact exquisitely arranged in proliferative rosettes (Fig 2E and F). This was striking because proliferative rosettes have never been reported in any of the previous Dicer mutant mouse lines (Kloosterman et al, 2006; De Pietri Tonelli et al, 2008). Figure 2. Formation of rosettes in the rostral telencephalon of Rx-Dicer mutants A. DAPI stain of sagittal sections through the rostral telencephalon of E17.5 control and Rx-Dicer mutant littermates showing the neocortex (NCx) and olfactory bulb (OB; dashed line). B. Expression pattern of Grm1 mRNA in OB (dotted line) of control and Rx-Dicer mutants. C. Quantification of OB perimeter (mean ± SEM; symbols indicate values for individual embryos); t-test, ***P < 0.001. N = 14 replicates per genotype. D. Immunostains of E17.5 control and Rx-Dicer mutant brains showing the distribution of progenitor cells (Ki67, red) and neurons (Tuj1, green). Arrowheads indicate rosettes. Sp, septum. E. Detail of a rosette from an E17.5 Rx-Dicer mutant embryo displaying typical features: closed apical surface (dotted line) with PH3+ apical mitoses (white arrowhead) and basal mitoses, surrounded by Tuj1+ neurons (green). Dashed line indicates the basal border of the rosette. F. Rostral half of an Rx-Dicer mutant E17.5 brain immunostained for Pax6, clarified, and segmented to reveal rosettes (yellow). C, caudal; R, rostral. G, H. BrdU incorporation and cell cycle re-entry analysis with the progenitor cell marker Ki67 at E17.5, in the rostral cortex of control embryos compared with rostral rosettes of Rx-Dicer mutants. Dotted lines indicate apical surface, and dashed lines indicate basal border of VZ. Arrowheads indicate double-positive cells. Data in histograms are mean ± SEM, symbols indicate values for individual embryos; n ≥ 3 embryos; chi-square test, *P < 0.05, ***P < 0.001. I. Apical lumen of rosettes immunostained against apical complex proteins (Par3, β-catenin), primary cilia (Arl13b), apical mitoses (PhVim), and neurons (Tuj1). J. Coronal section through the rostral telencephalon of an E14.5 Rx-Dicer mutant embryo illustrating the high abundance and location of rosettes (arrows), as revealed by the distribution of mitoses (PH3) and neurons (Tuj1). K–S. Analysis of the regional identity of rosettes in E14.5 Rx-Dicer mutants. (K) Schema of normal transcription factor expression patterns defining telencephalic regional identity. Expression of Ngn2, Tbr2, and Pax6 identifies rosettes in the rostro-dorsal telencephalon as having dorsal identity (L–N, P); Gsx2, Dlx2, and Dlx5 identify rosettes in the ventral telencephalon as having ventral identity (O, Q); Nkx2.1 identifies MGE rosettes as being normotopic (R); the absence of Gsx2 in LGE rosette cells identifies them as ectopic (S). Tuj1 labels neurons. In (L, M), arrowheads indicate the border between dorsal and ventral territories, and dotted line indicates the outer border of the telencephalon. LGE, lateral ganglionic eminence; MGE, medial ganglionic eminence; P, pallium; SP, subpallium. Data information: Scale bars, 500 μm (A, B, D, F), 25 μm (E, G, I, P–S), 200 μm (J–O). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Regionalized formation of rosettes in Dicer mutants and Irs2 overexpression A, B. Sagittal sections through the telencephalon of Rx-Dicer mutant embryos at the indicated ages stained for the indicated markers, showing the formation of proliferative rosettes selectively in the rostral but not the parietal (or caudal) regions, as indicated with brackets. C. Sagittal section through the telencephalon of an E14.5 wild-type embryo electroporated with Irs2 + Gfp encoding plasmids across the entire rostral and parietal regions of the neocortex, stained with the indicated markers. Details of each region are shown on the right. Arrows indicate proliferative rosettes, and arrowheads indicate ectopic Tuj1+ neurons in the apical border of the VZ. The electroporated parietal neocortex (NCx) remained unaffected, with perfect layering of neurons, and apical and basal mitoses. Data information: H, hippocampus; MGE, medial ganglionic eminence; OB, olfactory bulb; Spt, septum; Th, thalamus. Scale bars, 150 μm (A), 500 μm (B, C), 100 μm (C, details). Download figure Download PowerPoint Rosettes had a distinct laminar organization with apical–basal polarity, reminiscent of the telencephalic germinal layers in normal development. An inner lumen was delimited by a pseudostratified layer of progenitor cells undergoing apical mitosis, this was surrounded by a band of progenitor cells undergoing basal mitoses, and finally, these were enclosed by Tuj1+ neurons (Fig 2E). Brain clarification experiments showed numerous rosettes located in the rostral and ventral part of th
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