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• W4295346196 abstract "Article13 September 2022Open Access Source DataTransparent process Human-specific ARHGAP11B ensures human-like basal progenitor levels in hominid cerebral organoids Jan Fischer Jan Fischer Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: Conceptualization, Formal analysis, ​Investigation, Writing - original draft Search for more papers by this author Eduardo Fernández Ortuño Eduardo Fernández Ortuño orcid.org/0000-0001-9414-3392 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: Formal analysis, ​Investigation Search for more papers by this author Fabio Marsoner Fabio Marsoner Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, Mannheim, Germany Hector Institute for Translational Brain Research (HITBR gGmbH), Mannheim, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Contribution: Conceptualization, Formal analysis, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Annasara Artioli Annasara Artioli Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, Mannheim, Germany Hector Institute for Translational Brain Research (HITBR gGmbH), Mannheim, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Contribution: Formal analysis, ​Investigation Search for more papers by this author Jula Peters Jula Peters orcid.org/0000-0002-0770-8891 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: ​Investigation Search for more papers by this author Takashi Namba Takashi Namba Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Search for more papers by this author Christina Eugster Oegema Christina Eugster Oegema orcid.org/0000-0002-8895-0726 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: ​Investigation Search for more papers by this author Wieland B. Huttner Corresponding Author Wieland B. Huttner [email protected] orcid.org/0000-0003-4143-7201 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Julia Ladewig Corresponding Author Julia Ladewig [email protected] orcid.org/0000-0002-5943-7990 Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, Mannheim, Germany Hector Institute for Translational Brain Research (HITBR gGmbH), Mannheim, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Michael Heide Corresponding Author Michael Heide [email protected] [email protected] orcid.org/0000-0002-0752-8460 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Jan Fischer Jan Fischer Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: Conceptualization, Formal analysis, ​Investigation, Writing - original draft Search for more papers by this author Eduardo Fernández Ortuño Eduardo Fernández Ortuño orcid.org/0000-0001-9414-3392 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: Formal analysis, ​Investigation Search for more papers by this author Fabio Marsoner Fabio Marsoner Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, Mannheim, Germany Hector Institute for Translational Brain Research (HITBR gGmbH), Mannheim, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Contribution: Conceptualization, Formal analysis, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Annasara Artioli Annasara Artioli Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, Mannheim, Germany Hector Institute for Translational Brain Research (HITBR gGmbH), Mannheim, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Contribution: Formal analysis, ​Investigation Search for more papers by this author Jula Peters Jula Peters orcid.org/0000-0002-0770-8891 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: ​Investigation Search for more papers by this author Takashi Namba Takashi Namba Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Search for more papers by this author Christina Eugster Oegema Christina Eugster Oegema orcid.org/0000-0002-8895-0726 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: ​Investigation Search for more papers by this author Wieland B. Huttner Corresponding Author Wieland B. Huttner [email protected] orcid.org/0000-0003-4143-7201 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Julia Ladewig Corresponding Author Julia Ladewig [email protected] orcid.org/0000-0002-5943-7990 Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, Mannheim, Germany Hector Institute for Translational Brain Research (HITBR gGmbH), Mannheim, Germany German Cancer Research Center (DKFZ), Heidelberg, Germany Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Writing - review & editing Search for more papers by this author Michael Heide Corresponding Author Michael Heide [email protected] [email protected] orcid.org/0000-0002-0752-8460 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany Contribution: Conceptualization, Formal analysis, Supervision, Funding acquisition, ​Investigation, Writing - original draft, Writing - review & editing Search for more papers by this author Author Information Jan Fischer1,6,†, Eduardo Fernández Ortuño1,†, Fabio Marsoner2,3,4,†, Annasara Artioli2,3,4, Jula Peters1, Takashi Namba1,7, Christina Eugster Oegema1, Wieland B. Huttner *,1, Julia Ladewig *,2,3,4 and Michael Heide *,*,1,5 1Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, Dresden, Germany 2Central Institute of Mental Health, University of Heidelberg/Medical Faculty Mannheim, Mannheim, Germany 3Hector Institute for Translational Brain Research (HITBR gGmbH), Mannheim, Germany 4German Cancer Research Center (DKFZ), Heidelberg, Germany 5German Primate Center, Leibniz Institute for Primate Research, Göttingen, Germany 6Present address: Institute for Clinical Genetics, University Hospital Carl Gustav Carus, Dresden, Germany 7Present address: Neuroscience Center, HiLIFE - Helsinki Institute of Life Science, University of Helsinki, Helsinki, Finland † These authors contributed equally to this work *Corresponding author. Tel: +493512101500; E-mail: [email protected] *Corresponding author. Tel: +4962117036091; E-mail: [email protected] *Corresponding author (Lead contact). Tel: +495513851323; E-mail: [email protected]; [email protected] EMBO Reports (2022)23:e54728https://doi.org/10.15252/embr.202254728 See also: J Ding & AA Pollen (November 2022) PDFDownload PDF of article text and main figures.PDF PLUSDownload PDF of article text, main figures, expanded view figures and appendix. 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 The human-specific gene ARHGAP11B has been implicated in human neocortex expansion. However, the extent of ARHGAP11B's contribution to this expansion during hominid evolution is unknown. Here we address this issue by genetic manipulation of ARHGAP11B levels and function in chimpanzee and human cerebral organoids. ARHGAP11B expression in chimpanzee cerebral organoids doubles basal progenitor levels, the class of cortical progenitors with a key role in neocortex expansion. Conversely, interference with ARHGAP11B's function in human cerebral organoids decreases basal progenitors down to the chimpanzee level. Moreover, ARHGAP11A or ARHGAP11B rescue experiments in ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids indicate that lack of ARHGAP11B, but not of ARHGAP11A, decreases the abundance of basal radial glia—the basal progenitor type thought to be of particular relevance for neocortex expansion. Taken together, our findings demonstrate that ARHGAP11B is necessary and sufficient to ensure the elevated basal progenitor levels that characterize the fetal human neocortex, suggesting that this human-specific gene was a major contributor to neocortex expansion during human evolution. Synopsis Human and chimpanzee cerebral organoids reveal that human-specific ARHGAP11B is necessary and sufficient to ensure the elevated basal progenitor levels that characterize the fetal human neocortex. ARHGAP11B expression in chimpanzee cerebral organoids increases the abundance of cycling basal progenitors. Interference with ARHGAP11B's function in human cerebral organoids decreases the proliferation and abundance of basal progenitors down to the level of chimpanzee. ARHGAP11A or ARHGAP11B rescue experiments in ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids indicate that lack of ARHGAP11B, but not of ARHGAP11A, results in a massive decrease in the abundance of basal radial glia. Introduction The neocortex, the evolutionarily youngest part of the brain, is the seat of our higher cognitive abilities. It is therefore of crucial importance to investigate the development of the neocortex. This has been done in several model systems and has provided pivotal insight (Rakic, 2009; Lui et al, 2011; Florio & Huttner, 2014; Sun & Hevner, 2014; Dehay et al, 2015; Molnar et al, 2019; Silver et al, 2019). Identifying the features that characterize the development specifically of the human neocortex is, however, a fundamental challenge. The human neocortex exhibits an increase in size and in the numbers of neurons compared with non-human primates. This increase is thought to reflect a greater proliferative capacity of the cortical stem and progenitor cells (collectively referred to as cortical neural progenitor cells (cNPCs)) in human (Fish et al, 2008; Lui et al, 2011; Florio & Huttner, 2014; Sun & Hevner, 2014; Dehay et al, 2015). Over the past 8 years, genes have been identified that specifically evolved in the human lineage, that are preferentially expressed in cNPCs, and that promote cNPC proliferation (Florio et al, 2015, 2018; Fiddes et al, 2018; Suzuki et al, 2018). Such genes have therefore been implicated in human-specific features of neocortical development (Florio et al, 2015, 2018; Fiddes et al, 2018; Suzuki et al, 2018; Heide & Huttner, 2021). However, a human–chimpanzee comparison to explore whether such human-specific genes are responsible for a human-like cNPC proliferative capacity has not yet been carried out, mainly for the following reason. Whereas tissue of developing human neocortex can, in principle, be obtained and subjected to experimental studies, this is not the case for tissue of developing chimpanzee neocortex. A way out of this dilemma has been provided by recent, seminal advances in pluripotent stem cell (PSC) research, which led to the development of the brain organoid technology (Watanabe et al, 2005; Eiraku et al, 2008; Kadoshima et al, 2013; Lancaster et al, 2013, 2017; Pasca et al, 2015; Qian et al, 2016; Quadrato et al, 2017; Karzbrun et al, 2018; Giandomenico et al, 2019). A specific subtype of brain organoids, the cerebral organoids are relatively small (a few mm in diameter), three-dimensional (3D) structured cell assemblies that can be grown from embryonic stem cells (ESCs) (in the case of human) or induced pluripotent stem cells (iPSCs) (in the case of human and chimpanzee) and that emulate cerebral tissue (Lancaster et al, 2013, 2017; Kelava & Lancaster, 2016; Di Lullo & Kriegstein, 2017; Arlotta, 2018; Heide et al, 2018; Fischer et al, 2019). Thus, cerebral organoids have been shown to exhibit several (albeit not all) hallmarks of developing neocortical tissue, including a ventricular zone (VZ) and subventricular zone (SVZ) as well as the two major classes of cNPCs therein, the apical progenitors (APs) and the basal progenitors (BPs) (Kadoshima et al, 2013; Lancaster et al, 2013; Qian et al, 2016; Quadrato et al, 2017; Heide et al, 2018). Cerebral organoids also exhibit a cortical plate-like region with neuronal layers (NLs) containing the various types of cortical neurons (Kadoshima et al, 2013; Lancaster et al, 2013, 2017; Qian et al, 2016; Quadrato et al, 2017; Heide et al, 2018; Velasco et al, 2019). Moreover, human cerebral organoids have been shown to recapitulate gene expression programs of fetal human neocortex development (Camp et al, 2015; Velasco et al, 2019; Bhaduri et al, 2020). In light of these findings, cerebral organoids have emerged as a promising primate model system to study cortical development and evolution. In addition, cerebral organoids offer the opportunity of extrinsic genetic manipulation (Fischer et al, 2019). This is particularly relevant in the case of human-specific genes that in fetal human neocortex are preferentially expressed in cNPCs and hence have been implicated in human-specific features of neocortical development (Florio et al, 2015, 2018; Fiddes et al, 2018; Suzuki et al, 2018; Heide & Huttner, 2021). Examining such genes for their function in, and effects on, cNPC proliferation in cerebral organoids of human and chimpanzee, respectively, could not only provide corroborating evidence in support of their presumptive role in neocortical development during human evolution, but also provide further insights into their action and effects. ARHGAP11B is a human-specific gene (Sudmant et al, 2010; Dennis et al, 2017) and the first such gene to have been implicated in human neocortical development and evolution (Florio et al, 2015, 2016; Kalebic et al, 2018; Heide et al, 2020; Xing et al, 2021). In fetal human neocortex, ARHGAP11B is preferentially expressed in cNPCs (Florio et al, 2015, 2018). When (over)expressed in embryonic mouse and ferret neocortex, ARHGAP11B has been found to increase the proliferation and abundance of BPs (Florio et al, 2015; Kalebic et al, 2018; Xing et al, 2021), the cNPC class implicated in neocortical expansion during human development and evolution (Lui et al, 2011; Borrell & Götz, 2014; Florio & Huttner, 2014; Dehay et al, 2015). Moreover, a recent study in which ARHGAP11B was expressed under the control of its own promoter to physiological levels in the fetal neocortex of the common marmoset has demonstrated that this human-specific gene can indeed induce the hallmarks of neocortical expansion in this non-human primate, increasing neocortex size, folding, BP levels, and upper-layer neuron numbers (Heide et al, 2020). Consistent with this, physiological ARHGAP11B expression in a transgenic mouse line not only resulted in increased neocortical size and upper-layer neuron numbers that persist into adulthood, but also in increased cognitive abilities (Xing et al, 2021). Importantly, the ability of ARHGAP11B to increase the proliferation and abundance of BPs has been attributed not to the gene as it arose ≈ 5 mya by partial duplication of the widespread gene ARHGAP11A (Sudmant et al, 2010; Dennis et al, 2017), referred to as ancestral ARHGAP11B, but to an ARHGAP11B gene that subsequently underwent a point mutation, referred to as modern ARHGAP11B (Florio et al, 2016). These studies therefore establish (i) that modern ARHGAP11B is sufficient to expand BPs, including in primates, and (ii) that the resulting neocortex expansion and increase in upper-layer neuron numbers are associated with an increase in cognitive abilities. Considering these sets of findings together, the question arises to which extent ARHGAP11B contributes to the increase in cycling BPs in the context of the expansion of the neocortex in the course of human evolution. A first clue in this regard was obtained by the observation that a truncated form of the ARHGAP11A protein, ARHGAP11A220, which acts in a dominant-negative manner on ARHGAP11B's ability to amplify BPs in embryonic mouse neocortex, reduces the abundance of cycling BPs in fetal human neocortical tissue ex vivo (Namba et al, 2020). Yet, a key question regarding ARHGAP11B's role in human neocortex expansion remains unanswered: Can the human-specific ARHGAP11B gene increase the proliferation and abundance of BPs when expressed in cerebral organoids of the chimpanzee, our closest living relative? And, conversely, regarding human neocortical development: Is ARHGAP11B required to maintain the full level of BP proliferation and abundance in human cerebral organoids? In the present study, we have addressed these questions. In doing so, we provide support for the notion that ARHGAP11B is sufficient to increase BP proliferation and abundance to a human-like level in chimpanzee cerebral organoids. Conversely, we find that dominant-negative inhibition of ARHGAP11B's function by ARHGAP11A220 reduces cycling BP abundance in human cerebral organoids to the chimpanzee level. Finally, by subjecting ARHGAP11A plus ARHGAP11B double-knockout human forebrain organoids to either ARHGAP11A or ARHGAP11B rescue, we find that ARHGAP11B is essential to maintain the level of basal (or outer) radial glia (bRG), the BP type of particular relevance for neocortex expansion. Together, these findings provide direct evidence in support of an indispensable role of ARHGAP11B in neocortical expansion during human development and evolution. Results Human and chimpanzee cerebral organoids as a test system for gene function For most of the data presented in this study, human and chimpanzee cerebral organoids were grown from human iPSCs of the line SC102A1 (Camp et al, 2015; Mora-Bermudez et al, 2016; Kanton et al, 2019) and chimpanzee iPSCs of the line Sandra A (Mora-Bermudez et al, 2016; Kanton et al, 2019), respectively (for the iPSC line used to generate knockout forebrain organoids, see below). Cerebral organoids were generated according to an established protocol (Lancaster et al, 2013; Lancaster & Knoblich, 2014; Camp et al, 2015; Mora-Bermudez et al, 2016; Kanton et al, 2019). In brief, iPSCs were aggregated to form embryoid bodies followed by their transformation into 3D cerebral tissue exhibiting numerous ventricular structures (Fig 1A). Cerebral organoids were subjected to manipulations (see below) between day 51 and 55 (see Fig 1A). This time window was chosen based on the known time courses of VZ formation, SVZ formation, and deep- and upper-layer neuron generation. In contrast to macaque organoids, these time courses are roughly comparable between human and chimpanzee cerebral organoids (Mora-Bermudez et al, 2016; Otani et al, 2016; Kanton et al, 2019). After 51 or 55 days of organoid culture, various mixtures of DNA constructs, consisting of a cytosolic-GFP expression vector and either an expression vector with the cDNA of interest or the corresponding control vector, were then microinjected into the lumen of the larger ventricle-like structures within the cerebral organoids, followed by electroporation to transfect the cNPCs in the VZ (Fig 1A and B; Lancaster et al, 2013; Li et al, 2017; Fischer et al, 2019; Giandomenico et al, 2019). Depending on the specific scientific question asked, organoid culture was continued for 2–15 days after electroporation followed by fixation of the cerebral organoids, in the case of 2 days with addition of BrdU 1 h prior to fixation as indicated (Fig 1A). Fixed cerebral organoids were subjected to immunohistochemical analyses, using GFP immunofluorescence to identify the targeted cNPCs and their progeny (Fig 1A and B). These organoids were mainly of telencephalic identity as indicated by the expression of the telencephalic marker FOXG1 (Fig 1C). Figure 1. Experimental protocol of cerebral organoid production and time points of electroporation and analyses Timeline of cerebral organoid production detailing media as well as time points of electroporation (beginning of green bars), duration of vector expression (lengths of green bars; 2, 4, 10 and 15 days), and time points of fixation and analysis (end of green bars) of cerebral organoids. Left: Cartoon depicting the microinjection and electroporation of a ventricle-like structure of a cerebral organoid; Right: Immunofluorescence for GFP (green), combined with DAPI staining (white), of a 57-day-old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus control plasmid. Scale bar, 500 μm. Double immunofluorescence for GFP (green) and the telencephalic marker FOXG1 (yellow), combined with DAPI staining (white), of a 57-day-old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus control plasmid. Scale bar, 150 μm. Double immunofluorescence for SOX2 (magenta) and GFP (green), combined with DAPI staining (white), of a 57-day-old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus control plasmid (first row), of a 59-day-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus control plasmid (second row), and a 61-day-old chimpanzee cerebral organoid 10 days after electroporation with GFP expression plasmid plus control plasmid (third row). Tick marks indicate the border between VZ and SVZ/NL. Scale bars, 50 μm. Download figure Download PowerPoint Representative examples of control vector-transfected chimpanzee cerebral organoids 2, 4, and 10 days after electroporation are presented in Fig 1D and Appendix Figs S1–S3. These images show that depending on the time after electroporation, different cell populations in distinct zones of the developing cerebral organoid wall contain GFP-positive cells. This GFP expression reveals the transfected APs and their progeny and hence indicates the cells that, either directly or by inheritance, would be affected by a given electroporated DNA. Two days after electroporation, the majority of the GFP-positive cells was still observed in the VZ, colocalizing with a marker of proliferating cNPCs, SOX2 (Fig 1D). A minority of the GFP-positive cells was already observed basal to the VZ in the SVZ and neuronal layers (NL), colocalizing with a marker of basal progenitors, TBR2 (Englund et al, 2005; Sessa et al, 2008; Appendix Fig S1), but barely colocalizing with a marker of deep-layer neurons, CTIP2 (Arlotta et al, 2005; Molyneaux et al, 2007; Appendix Fig S2), and not colocalizing with a marker of upper-layer neurons, SATB2 (Alcamo et al, 2008; Britanova et al, 2008; Appendix Fig S3). These data are consistent with the length of the cell cycle of APs observed in chimpanzee cerebral organoids of ≈ 2 days (Mora-Bermudez et al, 2016) and suggest that the GFP-positive cells observed in the VZ 2 days after electroporation were either targeted APs, daughter APs of targeted APs, newborn BPs derived from targeted APs, or (few) newborn deep-layer neurons derived from targeted progenitors. Four days after electroporation, GFP-positive cells were observed in the basal region of the VZ, at the boundary between VZ and SVZ, and in the SVZ and NL, largely colocalizing with either SOX2 (Fig 1D), TBR2 (Appendix Fig S1) or CTIP2 (Appendix Fig S2) but not with SATB2 (Appendix Fig S3). This suggests that the GFP-positive cells observed in the VZ 4 days after electroporation were daughter APs of targeted APs, BPs derived from targeted APs, or newborn deep-layer neurons derived from targeted progenitors. Ten days after electroporation, GFP-positive cells were observed mostly in the basal SVZ and NL, colocalizing rarely with SOX2 (Fig 1D), still somewhat with TBR2 (Appendix Fig S1), mostly with CTIP2 (Appendix Fig S2), but not yet often with SATB2 (Appendix Fig S3). This is consistent with the notion that this longer period after electroporation should allow the targeted APs to carry out multiple rounds of BP-generating cell divisions, with the resulting BPs carrying out neuron-generating divisions. Accordingly, after the 10-day period following electroporation, a greater proportion of the targeted AP progeny is neurons, mostly of the deep-layer type, than after the 4-day period following electroporation (Appendix Figs S2 and S3). Expression of human-specific ARHGAP11B in chimpanzee cerebral organoids increases the abundance of cycling BPs Having established transfection of cerebral organoids as our test system, we first investigated whether, similar to the other non-human model systems of neocortex development previously used to study the effects of ARHGAP11B (Florio et al, 2015, 2016; Kalebic et al, 2018; Heide et al, 2020; Xing et al, 2021), ARHGAP11B would increase BP proliferation and abundance when expressed in chimpanzee cerebral organoids. For this purpose, we employed a previously used construct leading to ectopic expression of ARHGAP11B under the constitutive CAGGS promoter (pCAGGS-ARHGAP11B; Florio et al, 2015). (For details of this overexpression construct, and the justification and appropriateness of its use, please see Materials and Methods). We used the experimental protocol described above and in Fig 1 to determine the co-electroporation efficiency of the pCAGGS-EGFP and the pCAGGs-ARHGAP11B vectors in chimpanzee cerebral organoids. We found that ≥ 90% of the GFP-positive progeny of the targeted APs was also positive for ARHGAP11B by immunofluorescence at 2, 4, and 10 days after electroporation (Fig EV1A and B), indicating a high electroporation efficiency. Hence, we used this experimental protocol with a 2-day period between the electroporation of chimpanzee cerebral organoids with the ARHGAP11B expression vector and analysis of the transfected organoids by immunofluorescence for the BP marker TBR2. We found a marked, twofold increase in the proportion of the GFP-positive progeny of the targeted APs that were TBR2-positive, and hence newborn BPs, in the ARHGAP11B-transfected chimpanzee organoids in comparison to control-transfected organoids (Fig 2A and B). This increase by ≈ 10% points of the total GFP+ cells likely occurred at the expense of the APs, as ARHGAP11B has previously been shown to induce symmetric, consumptive BP-genic divisions of these cNPCs. These data therefore indicate that ARHGAP11B increases the abundance of BPs upon expression in chimpanzee cerebral organoids. Figure 2. Expression of ARHGAP11B in chimpanzee cerebral organoids increases the abundance of cycling BPs Double immunofluorescence for GFP (green) and the BP marker TBR2 (yellow), combined with DAPI staining (white), of a 57-day-old chimpanzee cerebral organoid 2 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+ and TBR2+ double-positive cells. Scale bars, 50 μm. Quantification of the proportion of GFP+ cells that are TBR2+ in 57-day-old chimpanzee cerebral organoids 2 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of nine control and nine ARHGAP11B-transfected cerebral organoids of two independent batches each; error bars indicate SD; ***P < 0.001 (two-sided Student's t-test). Triple immunofluorescence for GFP (green), the cycling cell marker Ki67 (magenta), and TBR2 (yellow), combined with DAPI staining (white), of a 59-day-old chimpanzee cerebral organoid 4 days after electroporation with GFP expression plasmid plus either control plasmid (top) or ARHGAP11B expression plasmid (bottom). Tick marks indicate the borders of the VZ and SVZ/NL; arrowheads indicate examples of GFP+, Ki67+ and TBR2+ triple-positive cells. Scale bars, 50 μm. Quantification of the proportion of GFP+ cells in the SVZ/NL that are Ki67+ and TBR2+ double-positive in 59-day-old chimpanzee cerebral organoids 4 days after electroporation with GFP expression plasmid plus either control plasmid (dark red) or ARHGAP11B expression plasmid (light red). Data are the mean of eight control and eight ARHGAP11B-transfected cerebral organoids of four independent batches each; error bars indicate SD; *P < 0.05 (one-sided Wilcoxon rank sum test). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. GFP and ARHGAP11B are co-expressed when co-electroporated in chimpanzee cerebral organoids Double immunofluorescence for GFP (green) and ARHGAP11B (magenta) of a 59-day-old chimpanzee cerebral or" @default.
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• W4295346196 title "Human‐specific <i> <scp>ARHGAP11B</scp> </i> ensures human‐like basal progenitor levels in hominid cerebral organoids" @default.
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• W4295346196 doi "https://doi.org/10.15252/embr.202254728" @default.
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