Differential sensitivity of structural, diffusion, and resting‐state functional MRI for detecting brain alterations and verbal memory impairment in temporal lobe epilepsy
Yu‐Hsuan ChangAnisa MarshallNaeim BahramiKushagra MathurSogol Stephanie JavadiAnny ReyesManu HegdeJerry J. ShihBrianna M. PaulDonald J. HaglerCarrie R. McDonald
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Abstract Objectives Temporal lobe epilepsy ( TLE ) is known to affect large‐scale gray and white matter networks, and these network changes likely contribute to the verbal memory impairments observed in many patients. In this study, we investigate multimodal imaging patterns of brain alterations in TLE and evaluate the sensitivity of different imaging measures to verbal memory impairment. Methods Diffusion tensor imaging ( DTI ), volumetric magnetic resonance imaging ( vMRI ), and resting‐state functional MRI (rs‐ fMRI ) were evaluated in 46 patients with TLE and 33 healthy controls to measure patterns of microstructural, structural, and functional alterations, respectively. These measurements were obtained within the white matter directly beneath neocortex (ie, superficial white matter [ SWM] ) for DTI and across neocortex for vMRI and rs‐ fMRI . The degree to which imaging alterations within left medial temporal lobe/posterior cingulate ( LMT / PC ) and left lateral temporal regions were associated with verbal memory performance was evaluated. Results Patients with left TLE and right TLE both demonstrated pronounced microstructural alterations (ie, decreased fractional anisotropy [ FA ] and increased mean diffusivity [ MD ]) spanning the entire frontal and temporolimbic SWM , which were highly lateralized to the ipsilateral hemisphere. Conversely, reductions in cortical thickness in vMRI and alterations in the magnitude of the rs‐ fMRI response were less pronounced and less lateralized than the microstructural changes. Both stepwise regression and mediation analyses further revealed that FA and MD within SWM in LMT / PC regions were the most robust predictors of verbal memory, and that these associations were independent of left hippocampal volume. Significance These findings suggest that microstructural loss within the SWM is pronounced in patients with TLE , and injury to the SWM within the LMT / PC region plays a critical role in verbal memory impairment.Keywords:
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The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans. ARHGAP11B is a human-specific gene that, based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex, has been proposed to have a key function in the evolutionary expansion of the neocortex. Here, we study the effects of ARHGAP11B expression in the developing neocortex of the gyrencephalic ferret. In contrast to its effects in mouse, ARHGAP11B markedly increases proliferative basal radial glia, a progenitor cell type thought to be instrumental for neocortical expansion, and results in extension of the neurogenic period and an increase in upper-layer neurons. Consequently, the postnatal ferret neocortex exhibits increased neuron density in the upper cortical layers and expands in both the radial and tangential dimensions. Thus, human-specific ARHGAP11B can elicit hallmarks of neocortical expansion in the developing ferret neocortex.
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An estimate of the total number of neurons in neocortex was obtained by multiplying an estimate of neocortical volume by the numerical density obtained with optical dissectors. Normal human brains were found to contain 20–25 × 109 neurons in neocortex. The overall numerical density of neurons in the neocortex was about 45–50 × 106/cm3 and did not show major variation with age or sex. Females have fewer neocortical neurons and correspondingly smaller cortices than males of the same age.
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Neocortex is an important part of the mammalian brain that is quite different from its homologue of the dorsal cortex in the reptilian brain. Whereas dorsal cortex is small, thin, and composed of a single layer of neurons, neocortex is thick and has six layers, while being variable across species in size, number of functional areas, and architectonic differentiation. Early mammals had little neocortex, with perhaps 20 areas of poor structural differentiation. Many extant mammals continue to have small brains with little neocortex, but they often have sensory specializations reflected in the organization of sensory areas in neocortex. In primates, neocortex is variously enlarged and characterized by structural and other specializations, including those of cortical networks devoted to vision and visuomotor processing. In humans, neocortex occupies 80% of the volume of the brain, where as many as 200 areas may exist.
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Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans. ARHGAP11B is a human-specific gene that, based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex, has been proposed to have a key function in the evolutionary expansion of the neocortex. Here, we study the effects of ARHGAP11B expression in the developing neocortex of the gyrencephalic ferret. In contrast to its effects in mouse, ARHGAP11B markedly increases proliferative basal radial glia, a progenitor cell type thought to be instrumental for neocortical expansion, and results in extension of the neurogenic period and an increase in upper-layer neurons. Consequently, the postnatal ferret neocortex exhibits increased neuron density in the upper cortical layers and expands in both the radial and tangential dimensions. Thus, human-specific ARHGAP11B can elicit hallmarks of neocortical expansion in the developing ferret neocortex. https://doi.org/10.7554/eLife.41241.001 eLife digest The human brain owes its characteristic wrinkled appearance to its outer layer, the cerebral cortex. All mammals have a cerebral cortex, but its size varies greatly between species. As the brain evolved, the neocortex, the evolutionarily youngest part of the cerebral cortex, expanded dramatically and so had to fold into wrinkles to fit inside the skull. The human neocortex is roughly three times bigger than that of our closest relatives, the chimpanzees, and helps support advanced cognitive skills such as reasoning and language. But how did the human neocortex become so big? The answer may lie in genes that are unique to humans, such as ARHGAP11B. Introducing ARHGAP11B into the neocortex of mouse embryos increases its size and can induce folding. It does this by increasing the number of neural progenitors, the cells that give rise to neurons. But there are two types of neural progenitors in mammalian neocortex: apical and basal. A subtype of the latter – basal radial glia – is thought to drive neocortex growth in human development. Unfortunately, mice have very few basal radial glia. This makes them unsuitable for testing whether ARHGAP11B acts via basal radial glia to enlarge the human neocortex. Kalebic et al. therefore introduced ARHGAP11B into ferret embryos in the womb. Ferrets have a larger neocortex than mice and possess more basal radial glia. Unlike in mice, introducing this gene into the ferret neocortex markedly increased the number of basal radial glia. It also extended the time window during which the basal radial glia produced neurons. These changes increased the number of neurons, particularly of a specific subtype found mainly in animals with large neocortex and thought to be involved in human cognition. Introducing human-specific ARHGAP11B into embryonic ferrets thus helped expand the ferret neocortex. This suggests that this gene may have a similar role in human brain development. Further experiments are needed to determine whether ferrets with the ARHGAP11B gene, and thus a larger neocortex, have enhanced cognitive abilities. If they do, testing these animals could provide insights into human cognition. The animals could also be used to model human brain diseases and to test potential treatments. https://doi.org/10.7554/eLife.41241.002 Introduction The expansion of the neocortex during primate evolution is thought to constitute one important basis for the unparalleled cognitive abilities of humans. The size of the neocortex is mainly regulated by the proliferative capacity of neural progenitor cells during cortical development and the length of the neurogenic period (Azevedo et al., 2009; Borrell and Götz, 2014; Dehay et al., 2015; Kaas, 2013; Kalebic et al., 2017; Krubitzer, 2007; Lui et al., 2011; Molnár et al., 2006; Rakic, 2009; Sousa et al., 2017; Wilsch-Bräuninger et al., 2016). Two major classes of neural progenitors can be distinguished: apical progenitors (APs), whose cell bodies reside in the ventricular zone (VZ), and basal progenitors (BPs), whose cell bodies reside in the subventricular zone (SVZ). Whereas APs are highly proliferative in the neocortex of all mammalian species studied (Götz and Huttner, 2005; Rakic, 2003a), BPs are highly proliferative only in species with an expanded neocortex (Borrell and Götz, 2014; Florio and Huttner, 2014; Lui et al., 2011; Reillo et al., 2011). Specifically, a subtype of BPs, called basal (or outer) radial glia (bRG), are thought to play a key role in the evolutionary expansion of the neocortex (Borrell and Götz, 2014; Florio and Huttner, 2014; Lui et al., 2011). Importantly, in species with an expanded neocortex, such as primates or the ferret, the SVZ has been shown to be divided into two distinct histological zones: the inner and outer SVZ (ISVZ and OSVZ, respectively) (Dehay et al., 2015; Reillo and Borrell, 2012; Smart et al., 2002). The OSVZ is uniquely important for the evolutionary expansion of the neocortex, as proliferative bRG are particularly abundant in this zone (Betizeau et al., 2013; Fietz et al., 2010; Hansen et al., 2010; Poluch and Juliano, 2015; Reillo and Borrell, 2012; Reillo et al., 2011; Smart et al., 2002). Increased proliferative capacity of bRG results in an amplification of BP number and is accompanied by a prolonged phase of production of late-born neurons (Geschwind and Rakic, 2013; Otani et al., 2016; Rakic, 2009). As the mammalian cerebral cortex is generated in an inside-out fashion, these late-born neurons occupy the upper-most layers of the cortex (Lodato and Arlotta, 2015; Molnár et al., 2006; Molyneaux et al., 2007; Rakic, 1972; Rakic, 2009; Sidman and Rakic, 1973). Thus, an increased generation of upper-layer neurons and increased thickness of the upper layers are also hallmarks of an expanded neocortex. The evolutionary expansion of the neocortex is characteristically accompanied by an increase in the abundance of proliferative bRG, in the length of the neurogenic period, and in the relative proportion of upper-layer neurons within the cortical plate (Borrell and Götz, 2014; Dehay et al., 2015; Florio and Huttner, 2014; Geschwind and Rakic, 2013; Lui et al., 2011; Molnár et al., 2006; Sousa et al., 2017; Wilsch-Bräuninger et al., 2016). This is most obvious when comparing extant rodents, such as mouse, with primates, such as human. Carnivores, such as ferret, display intermediate features (Borrell and Reillo, 2012; Hutsler et al., 2005; Kawasaki, 2014; Reillo et al., 2011). Specifically, ferrets exhibit a gyrified neocortex and, during development, a pronounced OSVZ populated with proliferative bRG (Barnette et al., 2009; Borrell and Reillo, 2012; Fietz et al., 2010; Kawasaki, 2014; Kawasaki et al., 2013; Poluch and Juliano, 2015; Reillo et al., 2011; Sawada and Watanabe, 2012; Smart and McSherry, 1986a;Smart and McSherry, 1986b ). In this context, it should be noted that in evolution, the split between the lineages leading to mouse and to human occurred a few million years later than that leading to ferret and human (Bininda-Emonds et al., 2007). In addition to the above-mentioned features associated with neocortex expansion in general, certain specific aspects of human neocortex expansion are thought to involve human-specific genomic changes. Recent transcriptomic studies established that certain previously identified human-specific genes (Bailey et al., 2002; Dennis and Eichler, 2016) are preferentially expressed in neural progenitor cells and have implicated these genes in human neocortex expansion (Fiddes et al., 2018; Florio et al., 2015; Florio et al., 2018; Florio et al., 2016; Suzuki et al., 2018). Among these genes, the one that showed the most specific expression in human bRG compared to neurons was ARHGAP11B (Florio et al., 2015). ARHGAP11B arose in evolution after the split of the human lineage from the chimpanzee lineage, as a product of a partial gene duplication of ARHGAP11A, a gene encoding a Rho GTPase activating protein (Dennis et al., 2017; Florio et al., 2015; Florio et al., 2016; Kagawa et al., 2013). Forced expression of ARHGAP11B in the embryonic mouse neocortex leads to an increase in BP proliferation and pool size (Florio et al., 2015). However, as described above, the mouse exhibits only a minute amount of bRG, a cell type thought to be instrumental for neocortex expansion, and the role of ARHGAP11B on the pool size of bRG, therefore, remains elusive. Additionally, the role of ARHGAP11B on the production of upper-layer neurons, another hallmark of the evolutionary expansion of the neocortex, is also unknown. Here, we study the effects of forced expression of ARHGAP11B in the developing ferret neocortex, which already exhibits several features of an expanded neocortex, including an abundance of bRG and of upper-layer neurons, and as such is a suitable model organism to address the role of ARHGAP11B in the evolutionary expansion of the neocortex. Results We expressed ARHGAP11B in the ferret neocortex starting at embryonic day 33 (E33), when both the generation of upper-layer neurons and formation of the OSVZ start (Martínez-Martínez et al., 2016). Specifically, we performed in utero electroporation of ferrets (Kawasaki et al., 2012; Kawasaki et al., 2013) at E33 with a plasmid encoding ARHGAP11B under the constitutive CAG promoter or an empty vector as control. The analyses of electroporated embryos were performed at four different developmental stages: E37, E40/postnatal day (P) 0, 10 and 16 (Figure 1—figure supplement 1A). To be able to visualize the electroporated area, we co-electroporated ARHGAP11B-expressing and control plasmids with vectors encoding fluorescent markers. For postnatal studies, to be able to distinguish the electroporated kits, the ARHGAP11B-expressing plasmid was co-electroporated with a GFP-encoding plasmid, and the control vector with an mCherry-encoding plasmid, or vice versa. For the sake of simplicity, we refer to both fluorescent markers as Fluorescent Protein (FP) from here onwards, and both FPs are depicted in green color in all figures. We detected ARHGAP11B transcript by RT-qPCR at all the stages analyzed and only in ferret embryos/kits subjected to ARHGAP11B in utero electroporation (Figure 1—figure supplement 1B–D). Additionally, immunofluorescence at E37 demonstrated the specific presence of the ARHGAP11B protein in neural progenitors of such embryos (Figure 1—figure supplement 1E,F and see Materials and methods for details). ARHGAP11B increases the abundance of BPs in the developing ferret neocortex We first examined the ability of ARHGAP11B to increase BP abundance in ferret. To this end, we immunostained E40/P0 ferret neocortex for PCNA, a marker of cycling cells, in order to identify progenitor cells (Figure 1A and Figure 1—figure supplement 2A). We observed an increase in the proportion of PCNA+ FP+ cells in OSVZ of the ARHGAP11B-expressing embryos compared to control (Figure 1B). The abundance of PCNA+ FP+ cells was increased in both ISVZ and OSVZ, but this increase was particularly strong in the OSVZ (Figure 1—figure supplement 2B). Of note, we did not detect any increase in the abundance of FP– progenitor cells in the SVZ, suggesting that ARHGAP11B does not promote any non-cell-autonomous effects (Figure 1—figure supplement 2C). Figure 1 with 2 supplements see all Download asset Open asset ARHGAP11B increases the abundance of BPs in the developing ferret neocortex. Ferret E33 neocortex was electroporated in utero with a plasmid encoding a fluorescent protein (FP) together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at E40/P0. (A) Double immunofluorescence for FP (green) and PCNA (magenta) (for the images of the single channels and DAPI staining, see Figure 1—figure supplement 2A). Images are single optical sections. Scale bars, 100 μm. Boxes (50 × 50 μm) indicate FP+ BPs in the OSVZ (1, top), ISVZ (2, middle) and VZ (3, bottom), shown at higher magnification in (A). (A) Dashed lines indicate a cell body contour. (B) Percentage of FP+ cells in the germinal zones (GZ total) and in the VZ, ISVZ and OSVZ that are PCNA+ upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 3 experiments. Error bars indicate SD; *, p <0.05; n.s., not statistically significant; Student's t-test. (C) Double immunofluorescence for FP (green) and phospho-vimentin (PhVim, magenta), combined with DAPI staining (white). Images are single optical sections. Scale bars, 50 μm. Vertical arrowheads, apical mitosis; horizontal arrowheads, basal mitosis. (D) Quantification of FP+ mitotic cells, as revealed by PhVim immunofluorescence, in a 200 µm-wide field of the cortical wall, upon control (white) and ARHGAP11B (black) electroporations. Apical, mitoses lining the ventricular surface; basal, mitoses away from the ventricle (Abv.VZ, abventricular VZ; ISVZ; OSVZ). Data are the mean of 4 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; n.s., not statistically significant; Student's t-test. (E) Mitotic bRG (single optical sections). Double immunofluorescence for FP (green) and phospho-vimentin (PhVim, magenta), combined with DAPI staining (white), upon electroporation of the plasmid encoding FP together with the plasmid encoding ARHGAP11B. Arrowheads, PhVim+ basal process of the mitotic bRG. Images are oriented with the apical side facing down and are 25 μm wide. (F) Quantification of mitotic bRG (FP+ PhVim+ cell bodies in the SVZ that contain a PhVim+ process), in a 200 µm-wide field of total SVZ (left), ISVZ (middle) and OSVZ (right), upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 4 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; Student's t-test. https://doi.org/10.7554/eLife.41241.003 We next immunostained the E40/P0 ferret neocortex for phospho-vimentin (PhVim), a marker of mitotic cells (Figure 1C). Our analysis revealed no effect of ARHGAP11B expression on apical mitoses (Figure 1D, left-most) and on basal mitoses in the abventricular VZ (Figure 1D, second column from left) compared to control. In contrast, a 3-fold increase in the abundance of basal mitotic cells in the SVZ was detected (Figure 1D, sum of ISVZ and OSVZ). This increase was observed for the ISVZ (2-fold, Figure 1D, second column from right), but was especially prominent for the OSVZ (5-fold, Figure 1D, right-most column). A comparably large increase (5-fold) was detected when examining mitotic bRG, that is, PhVim+ BPs exhibiting a PhVim+ process in the OSVZ (Figure 1E,F). bRG accounted for ≈50% of all BPs upon ARHGAP11B expression, and their relative proportion was not significantly changed compared to control or non-electroporated regions (Figure 1—figure supplement 2D). Of note, this strong increase in FP+ basal mitoses was not accompanied by any change in FP– mitotic cells (Figure 1—figure supplement 2E) nor by a change in thickness of the ferret germinal zones (Figure 1—figure supplement 2F). Taken together, these data indicate that ARHGAP11B markedly increases the abundance of BP, in particular bRG, when expressed in the embryonic ferret neocortex. ARHGAP11B increases the proportion of Sox2-positive bRG that are Tbr2-negative We next analyzed the ARHGAP11B-increased bRG in more detail. Proliferative neural progenitors, in particular apical radial glia (aRG) and bRG, characteristically express the transcription factor Sox2 (Pollen et al., 2015). We therefore immunostained E40/P0 ferret neocortex for Sox2 (Figure 2A) and detected a 40% increase in the proportion of Sox2+ FP+ cells in the germinal zones (GZs) (Figure 2B). This increase was exclusively due to an increase in BPs, as we observed a doubling of the proportion of Sox2+ FP+ cells in both the ISVZ and OSVZ, but no increase in the VZ (Figure 2C), upon ARHGAP11B expression. These data in turn are consistent with the effects of ARHGAP11B described above (Figure 1—figure supplement 2B). Figure 2 with 1 supplement see all Download asset Open asset ARHGAP11B increases the proportion of Sox2-positive bRG that are Tbr2-negative. Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by triple immunofluorescence for FP (green), Sox2 (yellow) and Tbr2 (magenta), combined with DAPI staining (white), at E40/P0. (A) Overview of the electroporated areas (single optical sections). Scale bars, 50 μm. (B, C) Percentage of FP+ cells in the germinal zones (B, GZ) and in the VZ (C, left), ISVZ (C, center) and OSVZ (C, right) that are Sox2+ upon control (white) and ARHGAP11B (black) electroporations. (D, E) Percentage of FP+ cells in the germinal zones (D, GZ) and in the VZ (E, left), ISVZ (E, center) and OSVZ (E, right) that are Tbr2+ upon control (white) and ARHGAP11B (black) electroporations. (F) Proliferative bRG (Sox2+ Tbr2– cell in the SVZ exhibiting radial morphology, single optical sections). Triple immunofluorescence for FP (green), Sox2 (yellow) and Tbr2 (magenta), combined with DAPI staining (white), upon electroporation of the plasmid encoding FP together with the plasmid encoding ARHGAP11B. Dashed lines, cell body; arrowheads, radial process. Images are oriented with the apical side facing down and are 25 μm wide. (G, H) Percentage of Sox2+ FP+ cells exhibiting radial morphology in the germinal zones (G, GZ) and in the VZ (H, left), ISVZ (H, center) and OSVZ (H, right) that are Tbr2– upon control (white) and ARHGAP11B (black) electroporations. (B–E, G, H) Data are the mean of 4 experiments. Error bars indicate SD; ***, p <0.001; **, p <0.01; *, p <0.05; n.s., not statistically significant; Student's t-test. https://doi.org/10.7554/eLife.41241.006 We then analyzed the FP+ cells for the expression of the transcription factor Tbr2 (Figure 2A), a marker of certain BPs (Englund et al., 2005). In embryonic mouse neocortex, Tbr2 is not only expressed in the predominant type of BP, the basal intermediate progenitors (bIPs) which are known to be neurogenic (Haubensak et al., 2004; Miyata et al., 2004; Noctor et al., 2004), but also (in contrast to an earlier report (Wang et al., 2011)) in the vast majority of mitotic bRG (Florio et al., 2015), which exhibit a low proliferative capacity (Wang et al., 2011). In contrast, human bRG, which exhibit a high proliferative capacity (Hansen et al., 2010; LaMonica et al., 2013), largely lack Tbr2 expression (Fietz et al., 2010; Hansen et al., 2010). Extrapolating from these data on mouse and human BPs to the bRG in embryonic ferret neocortex, many of which express Tbr2 (Reillo et al., 2011), it appears justified to assume that a portion of the latter bRG may be neurogenic and exhibit a reduced proliferative capacity. Consistent with this assumption and with the effects of ARHGAP11B expression on neural progenitors in the developing ferret neocortex described so far, we observed, upon expression of ARHGAP11B for 7 days, a decrease in the proportion of Tbr2+ FP+ progenitors in all GZs, which was statistically significant for the VZ (Figure 2D,E). In order to potentially obtain further cues as to the proliferative capacity of the ARHGAP11B-increased bRG in the developing ferret neocortex, we focused our attention on progenitor cells that (i) exhibited a radial morphology, (ii) expressed Sox2, but (iii) lacked Tbr2 expression (Figure 2F). Upon ARHGAP11B expression, we observed, in the sum of the GZs, a 20% increase in the proportion of radial Sox2+ cells that were Tbr2– (Figure 2G). This was largely due to an increase in the proportion of these cells in the OSVZ (Figure 2H), where more than 90% were Tbr2–. Considering that the ISVZ is the GZ with the highest amount of Tbr2+ BPs (Figure 2A), we examined the relative proportions of Sox2+ Trb2– and Sox2+ Tbr2+ BPs in the ISVZ and did not observe any significant difference in these proportions between control and ARHGAP11B expression (Figure 2—figure supplement 1). These findings are consistent with the notion that the ARHGAP11B-increased radial Sox2+ Tbr2– cells in the OSVZ are bRG. Studies in fetal human neocortex have established that such cells in the OSVZ are highly proliferative (Hansen et al., 2010 and LaMonica et al., 2013; for reviews see Florio and Huttner, 2014 and Lui et al., 2011). Hence, our finding that expression of ARHGAP11B in developing ferret neocortex results in a marked increase in the proportion of these cells suggests that this human-specific gene is sufficient to promote, in a gyrencephalic carnivore, the generation of bRG with putatively increased proliferative capacity. ARHGAP11B expression in developing ferret neocortex results in an extended neurogenic period We investigated the potential consequences of the ARHGAP11B-elicited increase in the abundance of BPs, notably of Sox2+ Tbr2– bRG, for neurogenesis in the developing ferret neocortex. To this end, we immunostained E40/P0 ferret neocortex for Tbr1, a transcription factor which is a marker of deep-layer neurons (Kolk et al., 2006) (Figure 3—figure supplement 1A), and for Satb2, a transcriptional regulator which is expressed in neurons that establish callosal projections and that are highly enriched in the upper layers of the cortical plate (CP) (Alcamo et al., 2008; Britanova et al., 2008) (Figure 3A). The vast majority (>90%) of the FP+ neurons in the CP of both control and ARHGAP11B-expressing ferret neocortex were found to be Satb2+ (Figure 3—figure supplement 1B). This high percentage is consistent with our experimental approach in which we targeted the embryonic ferret neural progenitors by in utero electroporation at E33, that is the time when the generation of the upper-layer neurons starts (Jackson et al., 1989; Martínez-Martínez et al., 2016). Figure 3 with 2 supplements see all Download asset Open asset ARHGAP11B expression in developing ferret neocortex results in an extended neurogenic period. Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP, together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at E40/P0 (A, B left), P10 (B center) and P16 (B right, (C–E). (A) Double immunofluorescence for FP (green) and Satb2 (magenta), combined with DAPI staining (white), of the E40/P0 ferret neocortex. The immunofluorescence of the same cryosection for Tbr1 is shown in Figure 3—figure supplement 1A. Images are single optical sections. Scale bars, 50 μm. (B) Distribution of Satb2+ FP+ neurons at E40/P0 (left), P10 (center) and P16 (right), between the cortical plate (CP, green) and germinal zones plus intermediate zone (GZ + IZ, yellow), upon control (Con, left) and ARHGAP11B (11B, right) electroporations. Data are the mean of 3 (P0 and P10) or 4 (P16) experiments. Error bars indicate SD; **, p <0.01; n.s., not statistically significant; two-way ANOVA with Bonferroni post-hoc tests (P10, Control CP vs. ARHGAP11B CP, p =0.0015). (C) Triple (immuno)fluorescence for FP (green), Satb2 (magenta) and EdU (yellow), combined with DAPI staining (white), of the P16 ferret neocortex, upon EdU injection at P5. Images are single optical sections. Scale bars, 1 mm. (C') Higher magnification of a FP+ Satb2+ EdU+ neuron upon electroporation of the plasmid encoding FP together with the plasmid encoding ARHGAP11B. Dashed lines, cell body. Images (single optical sections) are oriented with the apical side facing down and are 50 μm wide. (D) Percentage of FP+ cells that are EdU+ upon control (white) and ARHGAP11B (black) electroporations. (E) Percentage of EdU+ FP+ cells that are Satb2+ upon control (white) and ARHGAP11B (black) electroporations. (D, E) Data are the mean of 3 experiments. Error bars indicate SD; ***, p <0.001; *, p <0.05; Student's t-test. https://doi.org/10.7554/eLife.41241.008 Analysis of the distribution of Satb2+ FP+ neurons at E40/P0 between the CP on the one hand side, and the GZs plus the intermediate zone (IZ) on the other hand side, revealed that around 60% of the neurons had reached the CP in both control and ARHGAP11B-expressing ferret neocortex (Figure 3B left). We next examined electroporated ferret neocortex at P10 (Figure 3—figure supplement 2; analysis confined to gyri), which is the stage when neuron production is completed and neuron migration is terminating in the motor and somatosensory areas (Jackson et al., 1989; Smart and McSherry, 1986a; Smart and McSherry, 1986b). Consistent with this, our analysis of the control brains revealed that more than 90% of the Satb2+ FP+ neurons had reached the CP (Figure 3B middle). In contrast, only 70% of the Satb2+ FP+ neurons were found in the CP of the ARHGAP11B-expressing neocortex (Figure 3B middle). However, at P16, nearly all Satb2+ FP+ neurons were found in the CP in both control and ARHGAP11B-expressing neocortex (Figure 3B right; again, analysis confined to gyri). These data suggested that upon ARHGAP11B expression, either neurons migrate more slowly to the CP, or the neurogenic period is extended. To explore the latter scenario, we injected EdU into P5 ferret kits (i.e. 12 days after electroporation), which is the stage when neuronal progenitors undergo their very last neuron-generating cell divisions in the motor and somatosensory areas of the neocortex (Jackson et al., 1989; Smart and McSherry, 1986a; Smart and McSherry, 1986b). Analysis 11 days after EdU injection, at P16 (Figure 3C), revealed a 4-fold increase in the proportion of FP+ cells that were EdU+, in neocortical gyri of ARHGAP11B-expressing kits compared to control (Figure 3D). This was consistent with a prolonged, and hence increased, production of cells in ARHGAP11B-expressing kits, which in turn would be in line with the above described finding that ARHGAP11B increases the abundance of proliferative bRG. Importantly, among the EdU+ FP+ cells of the ARHGAP11B-expressing neocortex, 20% were Satb2+ neurons (Figure 3C' and E). In contrast, we did not detect a single Satb2+ EdU+ FP+ neuron in any of the control neocortices (Figure 3E). Collectively, these data indicate that neurogenesis in ARHGAP11B-expressing ferret neocortex continues longer than in control neocortex. ARHGAP11B expression in developing ferret neocortex results in a greater abundance of upper-layer neurons In light of the extension of the neurogenic period upon ARHGAP11B expression, we examined a potential increase in the abundance of the last-born neurons, that is, the upper-layer neurons. To this end, we first performed Nissl staining of the P16 ferret neocortex to visualize all neurons and the various layers of the CP, and immunostaining for Satb2, which is expressed in the majority of upper-layer neurons (Alcamo et al., 2008; Britanova et al., 2008; Lodato and Arlotta, 2015) (Figure 4A). These analyses, carried out on gyri, revealed (i) an increase in the abundance of FP+ cells in the CP (Figure 4—figure supplement 1A), and (ii) an alteration in the distribution of the FP+ cells between layers II-VI of the CP, with a greater proportion of cells in layers II-IV (Figure 4—figure supplement 1B), in the ARHGAP11B-expressing neocortex compared to control. A similar abundance increase (Figure 4—figure supplement 1C) and altered distribution (Figure 4B) were observed for Satb2+ FP+ neurons. Furthermore, the proportion of FP+ cells in the CP that were Satb2+ neurons was increased upon ARHGAP11B expression (Figure 4C). Figure 4 with 6 supplements see all Download asset Open asset ARHGAP11B expression results in a greater abundance of upper-layer neurons and expansion of the developing ferret neocortex. Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP, together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at P16. (A) Double immunofluorescence for FP (green) and Satb2 (magenta), combined with DAPI (white) and Nissl (yellow) staining, of the CP (single optical sections). Neuronal layers are marked on the left. Arrowheads, increased thickness of layer II upon ARHGAP11B expression. Scale bars, 200 μm. (B) Distribution of Satb2+ FP+ neurons between the neuronal layers upon control (Con, left) and ARHGAP11B (11B, right) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; ***, p <0.001; two-way ANOVA with Bonferroni post-hoc tests (Layer V, Control vs. ARHGAP11B, p <0.0001; Layer III, Control vs. ARHGAP11B, p =0.0073) (C) Percentage of FP+ cells in the CP that are Satb2+, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; ***, p <0.001; Student's t-test. (D) Percentage of FP+ cells in layers II + III that are Brn2+, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 5 experiments. Error bars indicate SD; *, p <0.05; Student's t-test. (E) Quantification of the gyrus thickness of control (Con) and ARHGAP11B-expressing (11B) ferret neocortex. Measurements were performed as described in Figure 4—figure supplement 3. All data are expressed as ratio between electroporated hemisphere (IUE) and non-electroporated contralateral hemisphere (non-IUE). Data are the mean (red lines) of 20 gyri per condition from six neocortices per condition. Error bars indicate SD; *, p <0.05; Student's t-test. (F) Quantification of layers II-IV thickness,
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Abstract Neocortex is the evolutionarily newest region in the brain, and is a structure with diversified size and morphology among mammalian species. Humans have the biggest neocortex compared to the body size, and their neocortex has many foldings, that is, gyri and sulci. Despite the recent methodological advances in in vitro models such as cerebral organoids, mice have been continuously used as a model system for studying human neocortical development because of the accessibility and practicality of in vivo gene manipulation. The commonly studied neocortical region, the lateral neocortex, generally recapitulates the developmental process of the human neocortex, however, there are several important factors missing in the lateral neocortex. First, basal (outer) radial glia (bRG), which are the main cell type providing the radial scaffold to the migrating neurons in the fetal human neocortex, are very few in the mouse lateral neocortex, thus the radial glial scaffold is different from the fetal human neocortex. Second, as a consequence of the difference in the radial glial scaffold, migrating neurons might exhibit different migratory behavior and thus distribution. To overcome those problems, we propose the mouse medial neocortex, where we have earlier revealed an abundance of bRG similar to the fetal human neocortex, as an alternative model system. We found that similar to the fetal human neocortex, the radial glial scaffold, neuronal migration and neuronal distribution are tangentially scattered in the mouse medial neocortex. Taken together, the embryonic mouse medial neocortex could be a suitable and accessible in vivo model system to study human neocortical development and its pathogenesis.
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The neocortex is a sheet of neurons organized in six layers, each receiving and projecting to specific brain areas depending on the neocortical region. This neuronal sheet, with some exceptions, displays very little horizontal anatomical segregation, but it is dynamically segregated into functional modules during stimulation. This chapter discusses the principal neurons of the neocortex, the interneurons, target selectivity in the neocortex, heterogeneity of synaptic dynamics, and alterations in disease.
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Article Figures and data Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans. ARHGAP11B is a human-specific gene that, based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex, has been proposed to have a key function in the evolutionary expansion of the neocortex. Here, we study the effects of ARHGAP11B expression in the developing neocortex of the gyrencephalic ferret. In contrast to its effects in mouse, ARHGAP11B markedly increases proliferative basal radial glia, a progenitor cell type thought to be instrumental for neocortical expansion, and results in extension of the neurogenic period and an increase in upper-layer neurons. Consequently, the postnatal ferret neocortex exhibits increased neuron density in the upper cortical layers and expands in both the radial and tangential dimensions. Thus, human-specific ARHGAP11B can elicit hallmarks of neocortical expansion in the developing ferret neocortex. https://doi.org/10.7554/eLife.41241.001 eLife digest The human brain owes its characteristic wrinkled appearance to its outer layer, the cerebral cortex. All mammals have a cerebral cortex, but its size varies greatly between species. As the brain evolved, the neocortex, the evolutionarily youngest part of the cerebral cortex, expanded dramatically and so had to fold into wrinkles to fit inside the skull. The human neocortex is roughly three times bigger than that of our closest relatives, the chimpanzees, and helps support advanced cognitive skills such as reasoning and language. But how did the human neocortex become so big? The answer may lie in genes that are unique to humans, such as ARHGAP11B. Introducing ARHGAP11B into the neocortex of mouse embryos increases its size and can induce folding. It does this by increasing the number of neural progenitors, the cells that give rise to neurons. But there are two types of neural progenitors in mammalian neocortex: apical and basal. A subtype of the latter – basal radial glia – is thought to drive neocortex growth in human development. Unfortunately, mice have very few basal radial glia. This makes them unsuitable for testing whether ARHGAP11B acts via basal radial glia to enlarge the human neocortex. Kalebic et al. therefore introduced ARHGAP11B into ferret embryos in the womb. Ferrets have a larger neocortex than mice and possess more basal radial glia. Unlike in mice, introducing this gene into the ferret neocortex markedly increased the number of basal radial glia. It also extended the time window during which the basal radial glia produced neurons. These changes increased the number of neurons, particularly of a specific subtype found mainly in animals with large neocortex and thought to be involved in human cognition. Introducing human-specific ARHGAP11B into embryonic ferrets thus helped expand the ferret neocortex. This suggests that this gene may have a similar role in human brain development. Further experiments are needed to determine whether ferrets with the ARHGAP11B gene, and thus a larger neocortex, have enhanced cognitive abilities. If they do, testing these animals could provide insights into human cognition. The animals could also be used to model human brain diseases and to test potential treatments. https://doi.org/10.7554/eLife.41241.002 Introduction The expansion of the neocortex during primate evolution is thought to constitute one important basis for the unparalleled cognitive abilities of humans. The size of the neocortex is mainly regulated by the proliferative capacity of neural progenitor cells during cortical development and the length of the neurogenic period (Azevedo et al., 2009; Borrell and Götz, 2014; Dehay et al., 2015; Kaas, 2013; Kalebic et al., 2017; Krubitzer, 2007; Lui et al., 2011; Molnár et al., 2006; Rakic, 2009; Sousa et al., 2017; Wilsch-Bräuninger et al., 2016). Two major classes of neural progenitors can be distinguished: apical progenitors (APs), whose cell bodies reside in the ventricular zone (VZ), and basal progenitors (BPs), whose cell bodies reside in the subventricular zone (SVZ). Whereas APs are highly proliferative in the neocortex of all mammalian species studied (Götz and Huttner, 2005; Rakic, 2003a), BPs are highly proliferative only in species with an expanded neocortex (Borrell and Götz, 2014; Florio and Huttner, 2014; Lui et al., 2011; Reillo et al., 2011). Specifically, a subtype of BPs, called basal (or outer) radial glia (bRG), are thought to play a key role in the evolutionary expansion of the neocortex (Borrell and Götz, 2014; Florio and Huttner, 2014; Lui et al., 2011). Importantly, in species with an expanded neocortex, such as primates or the ferret, the SVZ has been shown to be divided into two distinct histological zones: the inner and outer SVZ (ISVZ and OSVZ, respectively) (Dehay et al., 2015; Reillo and Borrell, 2012; Smart et al., 2002). The OSVZ is uniquely important for the evolutionary expansion of the neocortex, as proliferative bRG are particularly abundant in this zone (Betizeau et al., 2013; Fietz et al., 2010; Hansen et al., 2010; Poluch and Juliano, 2015; Reillo and Borrell, 2012; Reillo et al., 2011; Smart et al., 2002). Increased proliferative capacity of bRG results in an amplification of BP number and is accompanied by a prolonged phase of production of late-born neurons (Geschwind and Rakic, 2013; Otani et al., 2016; Rakic, 2009). As the mammalian cerebral cortex is generated in an inside-out fashion, these late-born neurons occupy the upper-most layers of the cortex (Lodato and Arlotta, 2015; Molnár et al., 2006; Molyneaux et al., 2007; Rakic, 1972; Rakic, 2009; Sidman and Rakic, 1973). Thus, an increased generation of upper-layer neurons and increased thickness of the upper layers are also hallmarks of an expanded neocortex. The evolutionary expansion of the neocortex is characteristically accompanied by an increase in the abundance of proliferative bRG, in the length of the neurogenic period, and in the relative proportion of upper-layer neurons within the cortical plate (Borrell and Götz, 2014; Dehay et al., 2015; Florio and Huttner, 2014; Geschwind and Rakic, 2013; Lui et al., 2011; Molnár et al., 2006; Sousa et al., 2017; Wilsch-Bräuninger et al., 2016). This is most obvious when comparing extant rodents, such as mouse, with primates, such as human. Carnivores, such as ferret, display intermediate features (Borrell and Reillo, 2012; Hutsler et al., 2005; Kawasaki, 2014; Reillo et al., 2011). Specifically, ferrets exhibit a gyrified neocortex and, during development, a pronounced OSVZ populated with proliferative bRG (Barnette et al., 2009; Borrell and Reillo, 2012; Fietz et al., 2010; Kawasaki, 2014; Kawasaki et al., 2013; Poluch and Juliano, 2015; Reillo et al., 2011; Sawada and Watanabe, 2012; Smart and McSherry, 1986a;Smart and McSherry, 1986b ). In this context, it should be noted that in evolution, the split between the lineages leading to mouse and to human occurred a few million years later than that leading to ferret and human (Bininda-Emonds et al., 2007). In addition to the above-mentioned features associated with neocortex expansion in general, certain specific aspects of human neocortex expansion are thought to involve human-specific genomic changes. Recent transcriptomic studies established that certain previously identified human-specific genes (Bailey et al., 2002; Dennis and Eichler, 2016) are preferentially expressed in neural progenitor cells and have implicated these genes in human neocortex expansion (Fiddes et al., 2018; Florio et al., 2015; Florio et al., 2018; Florio et al., 2016; Suzuki et al., 2018). Among these genes, the one that showed the most specific expression in human bRG compared to neurons was ARHGAP11B (Florio et al., 2015). ARHGAP11B arose in evolution after the split of the human lineage from the chimpanzee lineage, as a product of a partial gene duplication of ARHGAP11A, a gene encoding a Rho GTPase activating protein (Dennis et al., 2017; Florio et al., 2015; Florio et al., 2016; Kagawa et al., 2013). Forced expression of ARHGAP11B in the embryonic mouse neocortex leads to an increase in BP proliferation and pool size (Florio et al., 2015). However, as described above, the mouse exhibits only a minute amount of bRG, a cell type thought to be instrumental for neocortex expansion, and the role of ARHGAP11B on the pool size of bRG, therefore, remains elusive. Additionally, the role of ARHGAP11B on the production of upper-layer neurons, another hallmark of the evolutionary expansion of the neocortex, is also unknown. Here, we study the effects of forced expression of ARHGAP11B in the developing ferret neocortex, which already exhibits several features of an expanded neocortex, including an abundance of bRG and of upper-layer neurons, and as such is a suitable model organism to address the role of ARHGAP11B in the evolutionary expansion of the neocortex. Results We expressed ARHGAP11B in the ferret neocortex starting at embryonic day 33 (E33), when both the generation of upper-layer neurons and formation of the OSVZ start (Martínez-Martínez et al., 2016). Specifically, we performed in utero electroporation of ferrets (Kawasaki et al., 2012; Kawasaki et al., 2013) at E33 with a plasmid encoding ARHGAP11B under the constitutive CAG promoter or an empty vector as control. The analyses of electroporated embryos were performed at four different developmental stages: E37, E40/postnatal day (P) 0, 10 and 16 (Figure 1—figure supplement 1A). To be able to visualize the electroporated area, we co-electroporated ARHGAP11B-expressing and control plasmids with vectors encoding fluorescent markers. For postnatal studies, to be able to distinguish the electroporated kits, the ARHGAP11B-expressing plasmid was co-electroporated with a GFP-encoding plasmid, and the control vector with an mCherry-encoding plasmid, or vice versa. For the sake of simplicity, we refer to both fluorescent markers as Fluorescent Protein (FP) from here onwards, and both FPs are depicted in green color in all figures. We detected ARHGAP11B transcript by RT-qPCR at all the stages analyzed and only in ferret embryos/kits subjected to ARHGAP11B in utero electroporation (Figure 1—figure supplement 1B–D). Additionally, immunofluorescence at E37 demonstrated the specific presence of the ARHGAP11B protein in neural progenitors of such embryos (Figure 1—figure supplement 1E,F and see Materials and methods for details). ARHGAP11B increases the abundance of BPs in the developing ferret neocortex We first examined the ability of ARHGAP11B to increase BP abundance in ferret. To this end, we immunostained E40/P0 ferret neocortex for PCNA, a marker of cycling cells, in order to identify progenitor cells (Figure 1A and Figure 1—figure supplement 2A). We observed an increase in the proportion of PCNA+ FP+ cells in OSVZ of the ARHGAP11B-expressing embryos compared to control (Figure 1B). The abundance of PCNA+ FP+ cells was increased in both ISVZ and OSVZ, but this increase was particularly strong in the OSVZ (Figure 1—figure supplement 2B). Of note, we did not detect any increase in the abundance of FP– progenitor cells in the SVZ, suggesting that ARHGAP11B does not promote any non-cell-autonomous effects (Figure 1—figure supplement 2C). Figure 1 with 2 supplements see all Download asset Open asset ARHGAP11B increases the abundance of BPs in the developing ferret neocortex. Ferret E33 neocortex was electroporated in utero with a plasmid encoding a fluorescent protein (FP) together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at E40/P0. (A) Double immunofluorescence for FP (green) and PCNA (magenta) (for the images of the single channels and DAPI staining, see Figure 1—figure supplement 2A). Images are single optical sections. Scale bars, 100 μm. Boxes (50 × 50 μm) indicate FP+ BPs in the OSVZ (1, top), ISVZ (2, middle) and VZ (3, bottom), shown at higher magnification in (A). (A) Dashed lines indicate a cell body contour. (B) Percentage of FP+ cells in the germinal zones (GZ total) and in the VZ, ISVZ and OSVZ that are PCNA+ upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 3 experiments. Error bars indicate SD; *, p <0.05; n.s., not statistically significant; Student's t-test. (C) Double immunofluorescence for FP (green) and phospho-vimentin (PhVim, magenta), combined with DAPI staining (white). Images are single optical sections. Scale bars, 50 μm. Vertical arrowheads, apical mitosis; horizontal arrowheads, basal mitosis. (D) Quantification of FP+ mitotic cells, as revealed by PhVim immunofluorescence, in a 200 µm-wide field of the cortical wall, upon control (white) and ARHGAP11B (black) electroporations. Apical, mitoses lining the ventricular surface; basal, mitoses away from the ventricle (Abv.VZ, abventricular VZ; ISVZ; OSVZ). Data are the mean of 4 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; n.s., not statistically significant; Student's t-test. (E) Mitotic bRG (single optical sections). Double immunofluorescence for FP (green) and phospho-vimentin (PhVim, magenta), combined with DAPI staining (white), upon electroporation of the plasmid encoding FP together with the plasmid encoding ARHGAP11B. Arrowheads, PhVim+ basal process of the mitotic bRG. Images are oriented with the apical side facing down and are 25 μm wide. (F) Quantification of mitotic bRG (FP+ PhVim+ cell bodies in the SVZ that contain a PhVim+ process), in a 200 µm-wide field of total SVZ (left), ISVZ (middle) and OSVZ (right), upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 4 experiments. Error bars indicate SD; **, p <0.01; *, p <0.05; Student's t-test. https://doi.org/10.7554/eLife.41241.003 We next immunostained the E40/P0 ferret neocortex for phospho-vimentin (PhVim), a marker of mitotic cells (Figure 1C). Our analysis revealed no effect of ARHGAP11B expression on apical mitoses (Figure 1D, left-most) and on basal mitoses in the abventricular VZ (Figure 1D, second column from left) compared to control. In contrast, a 3-fold increase in the abundance of basal mitotic cells in the SVZ was detected (Figure 1D, sum of ISVZ and OSVZ). This increase was observed for the ISVZ (2-fold, Figure 1D, second column from right), but was especially prominent for the OSVZ (5-fold, Figure 1D, right-most column). A comparably large increase (5-fold) was detected when examining mitotic bRG, that is, PhVim+ BPs exhibiting a PhVim+ process in the OSVZ (Figure 1E,F). bRG accounted for ≈50% of all BPs upon ARHGAP11B expression, and their relative proportion was not significantly changed compared to control or non-electroporated regions (Figure 1—figure supplement 2D). Of note, this strong increase in FP+ basal mitoses was not accompanied by any change in FP– mitotic cells (Figure 1—figure supplement 2E) nor by a change in thickness of the ferret germinal zones (Figure 1—figure supplement 2F). Taken together, these data indicate that ARHGAP11B markedly increases the abundance of BP, in particular bRG, when expressed in the embryonic ferret neocortex. ARHGAP11B increases the proportion of Sox2-positive bRG that are Tbr2-negative We next analyzed the ARHGAP11B-increased bRG in more detail. Proliferative neural progenitors, in particular apical radial glia (aRG) and bRG, characteristically express the transcription factor Sox2 (Pollen et al., 2015). We therefore immunostained E40/P0 ferret neocortex for Sox2 (Figure 2A) and detected a 40% increase in the proportion of Sox2+ FP+ cells in the germinal zones (GZs) (Figure 2B). This increase was exclusively due to an increase in BPs, as we observed a doubling of the proportion of Sox2+ FP+ cells in both the ISVZ and OSVZ, but no increase in the VZ (Figure 2C), upon ARHGAP11B expression. These data in turn are consistent with the effects of ARHGAP11B described above (Figure 1—figure supplement 2B). Figure 2 with 1 supplement see all Download asset Open asset ARHGAP11B increases the proportion of Sox2-positive bRG that are Tbr2-negative. Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by triple immunofluorescence for FP (green), Sox2 (yellow) and Tbr2 (magenta), combined with DAPI staining (white), at E40/P0. (A) Overview of the electroporated areas (single optical sections). Scale bars, 50 μm. (B, C) Percentage of FP+ cells in the germinal zones (B, GZ) and in the VZ (C, left), ISVZ (C, center) and OSVZ (C, right) that are Sox2+ upon control (white) and ARHGAP11B (black) electroporations. (D, E) Percentage of FP+ cells in the germinal zones (D, GZ) and in the VZ (E, left), ISVZ (E, center) and OSVZ (E, right) that are Tbr2+ upon control (white) and ARHGAP11B (black) electroporations. (F) Proliferative bRG (Sox2+ Tbr2– cell in the SVZ exhibiting radial morphology, single optical sections). Triple immunofluorescence for FP (green), Sox2 (yellow) and Tbr2 (magenta), combined with DAPI staining (white), upon electroporation of the plasmid encoding FP together with the plasmid encoding ARHGAP11B. Dashed lines, cell body; arrowheads, radial process. Images are oriented with the apical side facing down and are 25 μm wide. (G, H) Percentage of Sox2+ FP+ cells exhibiting radial morphology in the germinal zones (G, GZ) and in the VZ (H, left), ISVZ (H, center) and OSVZ (H, right) that are Tbr2– upon control (white) and ARHGAP11B (black) electroporations. (B–E, G, H) Data are the mean of 4 experiments. Error bars indicate SD; ***, p <0.001; **, p <0.01; *, p <0.05; n.s., not statistically significant; Student's t-test. https://doi.org/10.7554/eLife.41241.006 We then analyzed the FP+ cells for the expression of the transcription factor Tbr2 (Figure 2A), a marker of certain BPs (Englund et al., 2005). In embryonic mouse neocortex, Tbr2 is not only expressed in the predominant type of BP, the basal intermediate progenitors (bIPs) which are known to be neurogenic (Haubensak et al., 2004; Miyata et al., 2004; Noctor et al., 2004), but also (in contrast to an earlier report (Wang et al., 2011)) in the vast majority of mitotic bRG (Florio et al., 2015), which exhibit a low proliferative capacity (Wang et al., 2011). In contrast, human bRG, which exhibit a high proliferative capacity (Hansen et al., 2010; LaMonica et al., 2013), largely lack Tbr2 expression (Fietz et al., 2010; Hansen et al., 2010). Extrapolating from these data on mouse and human BPs to the bRG in embryonic ferret neocortex, many of which express Tbr2 (Reillo et al., 2011), it appears justified to assume that a portion of the latter bRG may be neurogenic and exhibit a reduced proliferative capacity. Consistent with this assumption and with the effects of ARHGAP11B expression on neural progenitors in the developing ferret neocortex described so far, we observed, upon expression of ARHGAP11B for 7 days, a decrease in the proportion of Tbr2+ FP+ progenitors in all GZs, which was statistically significant for the VZ (Figure 2D,E). In order to potentially obtain further cues as to the proliferative capacity of the ARHGAP11B-increased bRG in the developing ferret neocortex, we focused our attention on progenitor cells that (i) exhibited a radial morphology, (ii) expressed Sox2, but (iii) lacked Tbr2 expression (Figure 2F). Upon ARHGAP11B expression, we observed, in the sum of the GZs, a 20% increase in the proportion of radial Sox2+ cells that were Tbr2– (Figure 2G). This was largely due to an increase in the proportion of these cells in the OSVZ (Figure 2H), where more than 90% were Tbr2–. Considering that the ISVZ is the GZ with the highest amount of Tbr2+ BPs (Figure 2A), we examined the relative proportions of Sox2+ Trb2– and Sox2+ Tbr2+ BPs in the ISVZ and did not observe any significant difference in these proportions between control and ARHGAP11B expression (Figure 2—figure supplement 1). These findings are consistent with the notion that the ARHGAP11B-increased radial Sox2+ Tbr2– cells in the OSVZ are bRG. Studies in fetal human neocortex have established that such cells in the OSVZ are highly proliferative (Hansen et al., 2010 and LaMonica et al., 2013; for reviews see Florio and Huttner, 2014 and Lui et al., 2011). Hence, our finding that expression of ARHGAP11B in developing ferret neocortex results in a marked increase in the proportion of these cells suggests that this human-specific gene is sufficient to promote, in a gyrencephalic carnivore, the generation of bRG with putatively increased proliferative capacity. ARHGAP11B expression in developing ferret neocortex results in an extended neurogenic period We investigated the potential consequences of the ARHGAP11B-elicited increase in the abundance of BPs, notably of Sox2+ Tbr2– bRG, for neurogenesis in the developing ferret neocortex. To this end, we immunostained E40/P0 ferret neocortex for Tbr1, a transcription factor which is a marker of deep-layer neurons (Kolk et al., 2006) (Figure 3—figure supplement 1A), and for Satb2, a transcriptional regulator which is expressed in neurons that establish callosal projections and that are highly enriched in the upper layers of the cortical plate (CP) (Alcamo et al., 2008; Britanova et al., 2008) (Figure 3A). The vast majority (>90%) of the FP+ neurons in the CP of both control and ARHGAP11B-expressing ferret neocortex were found to be Satb2+ (Figure 3—figure supplement 1B). This high percentage is consistent with our experimental approach in which we targeted the embryonic ferret neural progenitors by in utero electroporation at E33, that is the time when the generation of the upper-layer neurons starts (Jackson et al., 1989; Martínez-Martínez et al., 2016). Figure 3 with 2 supplements see all Download asset Open asset ARHGAP11B expression in developing ferret neocortex results in an extended neurogenic period. Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP, together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at E40/P0 (A, B left), P10 (B center) and P16 (B right, (C–E). (A) Double immunofluorescence for FP (green) and Satb2 (magenta), combined with DAPI staining (white), of the E40/P0 ferret neocortex. The immunofluorescence of the same cryosection for Tbr1 is shown in Figure 3—figure supplement 1A. Images are single optical sections. Scale bars, 50 μm. (B) Distribution of Satb2+ FP+ neurons at E40/P0 (left), P10 (center) and P16 (right), between the cortical plate (CP, green) and germinal zones plus intermediate zone (GZ + IZ, yellow), upon control (Con, left) and ARHGAP11B (11B, right) electroporations. Data are the mean of 3 (P0 and P10) or 4 (P16) experiments. Error bars indicate SD; **, p <0.01; n.s., not statistically significant; two-way ANOVA with Bonferroni post-hoc tests (P10, Control CP vs. ARHGAP11B CP, p =0.0015). (C) Triple (immuno)fluorescence for FP (green), Satb2 (magenta) and EdU (yellow), combined with DAPI staining (white), of the P16 ferret neocortex, upon EdU injection at P5. Images are single optical sections. Scale bars, 1 mm. (C') Higher magnification of a FP+ Satb2+ EdU+ neuron upon electroporation of the plasmid encoding FP together with the plasmid encoding ARHGAP11B. Dashed lines, cell body. Images (single optical sections) are oriented with the apical side facing down and are 50 μm wide. (D) Percentage of FP+ cells that are EdU+ upon control (white) and ARHGAP11B (black) electroporations. (E) Percentage of EdU+ FP+ cells that are Satb2+ upon control (white) and ARHGAP11B (black) electroporations. (D, E) Data are the mean of 3 experiments. Error bars indicate SD; ***, p <0.001; *, p <0.05; Student's t-test. https://doi.org/10.7554/eLife.41241.008 Analysis of the distribution of Satb2+ FP+ neurons at E40/P0 between the CP on the one hand side, and the GZs plus the intermediate zone (IZ) on the other hand side, revealed that around 60% of the neurons had reached the CP in both control and ARHGAP11B-expressing ferret neocortex (Figure 3B left). We next examined electroporated ferret neocortex at P10 (Figure 3—figure supplement 2; analysis confined to gyri), which is the stage when neuron production is completed and neuron migration is terminating in the motor and somatosensory areas (Jackson et al., 1989; Smart and McSherry, 1986a; Smart and McSherry, 1986b). Consistent with this, our analysis of the control brains revealed that more than 90% of the Satb2+ FP+ neurons had reached the CP (Figure 3B middle). In contrast, only 70% of the Satb2+ FP+ neurons were found in the CP of the ARHGAP11B-expressing neocortex (Figure 3B middle). However, at P16, nearly all Satb2+ FP+ neurons were found in the CP in both control and ARHGAP11B-expressing neocortex (Figure 3B right; again, analysis confined to gyri). These data suggested that upon ARHGAP11B expression, either neurons migrate more slowly to the CP, or the neurogenic period is extended. To explore the latter scenario, we injected EdU into P5 ferret kits (i.e. 12 days after electroporation), which is the stage when neuronal progenitors undergo their very last neuron-generating cell divisions in the motor and somatosensory areas of the neocortex (Jackson et al., 1989; Smart and McSherry, 1986a; Smart and McSherry, 1986b). Analysis 11 days after EdU injection, at P16 (Figure 3C), revealed a 4-fold increase in the proportion of FP+ cells that were EdU+, in neocortical gyri of ARHGAP11B-expressing kits compared to control (Figure 3D). This was consistent with a prolonged, and hence increased, production of cells in ARHGAP11B-expressing kits, which in turn would be in line with the above described finding that ARHGAP11B increases the abundance of proliferative bRG. Importantly, among the EdU+ FP+ cells of the ARHGAP11B-expressing neocortex, 20% were Satb2+ neurons (Figure 3C' and E). In contrast, we did not detect a single Satb2+ EdU+ FP+ neuron in any of the control neocortices (Figure 3E). Collectively, these data indicate that neurogenesis in ARHGAP11B-expressing ferret neocortex continues longer than in control neocortex. ARHGAP11B expression in developing ferret neocortex results in a greater abundance of upper-layer neurons In light of the extension of the neurogenic period upon ARHGAP11B expression, we examined a potential increase in the abundance of the last-born neurons, that is, the upper-layer neurons. To this end, we first performed Nissl staining of the P16 ferret neocortex to visualize all neurons and the various layers of the CP, and immunostaining for Satb2, which is expressed in the majority of upper-layer neurons (Alcamo et al., 2008; Britanova et al., 2008; Lodato and Arlotta, 2015) (Figure 4A). These analyses, carried out on gyri, revealed (i) an increase in the abundance of FP+ cells in the CP (Figure 4—figure supplement 1A), and (ii) an alteration in the distribution of the FP+ cells between layers II-VI of the CP, with a greater proportion of cells in layers II-IV (Figure 4—figure supplement 1B), in the ARHGAP11B-expressing neocortex compared to control. A similar abundance increase (Figure 4—figure supplement 1C) and altered distribution (Figure 4B) were observed for Satb2+ FP+ neurons. Furthermore, the proportion of FP+ cells in the CP that were Satb2+ neurons was increased upon ARHGAP11B expression (Figure 4C). Figure 4 with 6 supplements see all Download asset Open asset ARHGAP11B expression results in a greater abundance of upper-layer neurons and expansion of the developing ferret neocortex. Ferret E33 neocortex was electroporated in utero with a plasmid encoding FP, together with either a plasmid encoding ARHGAP11B or empty vector (Control), followed by analysis at P16. (A) Double immunofluorescence for FP (green) and Satb2 (magenta), combined with DAPI (white) and Nissl (yellow) staining, of the CP (single optical sections). Neuronal layers are marked on the left. Arrowheads, increased thickness of layer II upon ARHGAP11B expression. Scale bars, 200 μm. (B) Distribution of Satb2+ FP+ neurons between the neuronal layers upon control (Con, left) and ARHGAP11B (11B, right) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; ***, p <0.001; two-way ANOVA with Bonferroni post-hoc tests (Layer V, Control vs. ARHGAP11B, p <0.0001; Layer III, Control vs. ARHGAP11B, p =0.0073) (C) Percentage of FP+ cells in the CP that are Satb2+, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 6 experiments. Error bars indicate SD; ***, p <0.001; Student's t-test. (D) Percentage of FP+ cells in layers II + III that are Brn2+, upon control (white) and ARHGAP11B (black) electroporations. Data are the mean of 5 experiments. Error bars indicate SD; *, p <0.05; Student's t-test. (E) Quantification of the gyrus thickness of control (Con) and ARHGAP11B-expressing (11B) ferret neocortex. Measurements were performed as described in Figure 4—figure supplement 3. All data are expressed as ratio between electroporated hemisphere (IUE) and non-electroporated contralateral hemisphere (non-IUE). Data are the mean (red lines) of 20 gyri per condition from six neocortices per condition. Error bars indicate SD; *, p <0.05; Student's t-test. (F) Quantification of layers II-IV thickness,
Neocortex
Human brain
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Abstract The mammalian cerebral neocortex occupies the largest area of the cerebral cortex and is cytoarchitectually composed of six layers (I–VI). Recent molecular analysis has begun to reveal the existence of various developmental programs, including the genetic regulation of arealization of the neocortex. Although an increasing number of molecular determinants of the developmental stages of the neocortex have been identified, no genes specifically expressed in the adult neocortex have been identified to date. By global screening using microarrays, combined with systematic in situ hybridization, we identified a zinc‐finger type transcription factor, Fez1, which is expressed predominantly in the mouse adult neocortex. No other genes in the neocortex have been shown to date to have their expression with such high specificity. Using two‐color in situ hybridization, we show that Fez1 is mainly expressed in cortical layers V and VI, not in γ‐aminobutyric acid neurons but in pyramidal neurons, the projection neurons of the cerebral cortex. Immunohistochemistry also shows that Fez1 is expressed in deep layers of the neocortex. Fez1 will be invaluable not only for the molecular understanding of corticogenesis but also for understanding the physiological functions of the adult neocortex, as well as for the use of its promoter in gene‐manipulated animals and in conditional expression systems.
Neocortex
Corticogenesis
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Abstract The evolutionary increase in size and complexity of the primate neocortex is thought to underlie the higher cognitive abilities of humans. ARHGAP11B is a human-specific gene that, based on its expression pattern in fetal human neocortex and progenitor effects in embryonic mouse neocortex, has been proposed to have a key function in the evolutionary expansion of the neocortex. Here, we study the effects of ARHGAP11B expression in the developing neocortex of the gyrencephalic ferret. In contrast to its effects in mouse, ARHGAP11B markedly increases proliferative basal radial glia, a progenitor cell type thought to be instrumental for neocortical expansion, and results in extension of the neurogenic period and an increase in upper-layer neurons. As a consequence, the postnatal ferret neocortex exhibits an increased neuron density in the upper cortical layers and expands in the radial dimension. Thus, human-specific ARHGAP11B can elicit hallmarks of neocortical expansion in developing ferret neocortex.
Neocortex
Progenitor
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