Regulation of temporal properties of neural stem cells and transition timing of neurogenesis and gliogenesis during mammalian neocortical development

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Review

Regulation of temporal properties of neural stem cells and transition timing of neurogenesis and gliogenesis during mammalian neocortical development ⁎

Toshiyuki Ohtsukaa,b,c, , Ryoichiro Kageyamaa,b,c,d a

Institute for Frontier Life and Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan Kyoto University Graduate School of Medicine, Kyoto, 606-8501, Japan c Kyoto University Graduate School of Biostudies, Kyoto, 606-8501, Japan d Institute for Integrated Cell-Material Sciences (iCeMS), Kyoto University, Kyoto, 606-8501, Japan b

A R T I C LE I N FO

A B S T R A C T

Keywords: Neural stem cell Hbp1 Hes5 Neurogenesis Gliogenesis Neocortical development

In the developing mammalian neocortex, neural stem cells (NSCs) gradually alter their characteristics as development proceeds. NSCs initially expand the progenitor pool by symmetric proliferative division and then shift to asymmetric neurogenic division to commence neurogenesis. NSCs sequentially give rise to deep layer neurons first and superficial layer neurons later through mid- to late-embryonic stages, followed by shifting to a gliogenic phase at perinatal stages. The precise mechanisms regulating developmental timing of the transition from symmetric to asymmetric division have not been fully elucidated; however, gradual elongation in cell cycle length and concomitant accumulation of determinants that promote neuronal differentiation may function as a biological clock that regulates the onset of asymmetric neurogenic division. On the other hand, epigenetic regulatory systems have been implicated in the regulation of transition timing of neurogenesis and gliogenesis; the polycomb group (PcG) complex and Hmga genes have been found to govern the developmental timing by modulating chromatin structure during neocortical development. Furthermore, we uncovered several factors and mechanisms underlying the regulation of timing of neocortical neurogenesis and gliogenesis. In this review, we discuss recent findings regarding the mechanisms that govern the temporal properties of NSCs and the precise transition timing during neocortical development.

1. Introduction During mammalian neocortical development, neural stem cells (NSCs) gradually alter their morphology and characteristics to give rise to distinct cell types in a precise temporal order [1–3]. NSCs transform from neuroepithelial cells to radial glial cells (RGCs) and finally remain as astrocyte-like cells in the postnatal and adult brain (Doetsch et al., 1999; Noctor et al., 2001; Noctor et al., 2002) [4–6]. Neuroepithelial cells initially divide symmetrically to multiply their copies and exponentially expand the stem cell pool, after which division mode switches from symmetric to asymmetric and NSCs commence neurogenesis (Takahashi et al., 1994; Chenn et al., 1995) [7,8]. After the onset of neurogenesis, NSCs transform to RGCs and sequentially produce deep layer neurons first and then superficial layer neurons by asymmetric neurogenic divisions (Noctor et al., 2001; Noctor et al., 2002) [5,6]. After the neurogenic period is over, gliogenesis supersedes neurogenesis at late embryonic or perinatal stages. Hence, the timing of transition from symmetric proliferative to asymmetric neurogenic

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division is particularly important in determining onset of neurogenesis and size of the initial stem cell pool. On the other hand, the timing of transition from deep to superficial layer neurogenesis and from neurogenesis to gliogenesis is crucial in determining the total number of neocortical neurons and the numbers of each layer-specific neuronal population. Although these transition timings are critical in determining the eventual size and morphology of the neocortex, the precise regulatory mechanisms have not been fully elucidated. In this review, we discuss recent findings regarding the temporal alterations in characteristics of NSCs and the mechanisms that govern precise transition timing of neurogenesis and gliogenesis during mammalian neocortical development. 2. Temporal properties of neocortical NSCs NSCs gradually alter their capacity to proliferate or produce various types of neural cells in a temporally regulated manner (Temple, 2001)

Corresponding author at: Institute for Frontier Life and Medical Sciences, Kyoto University, 53 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. E-mail address: [email protected] (T. Ohtsuka).

https://doi.org/10.1016/j.semcdb.2019.01.007 Received 7 September 2018; Received in revised form 5 December 2018; Accepted 8 January 2019 1084-9521/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Ohtsuka, T., Seminars in Cell and Developmental Biology, https://doi.org/10.1016/j.semcdb.2019.01.007

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These results demonstrate that both Tcf7l1 and Klf15 are involved in the maintenance of NSCs by inhibiting premature neuronal differentiation. Based on their temporal expression patterns, it is likely that Tcf7l1 and Klf15 play critical roles in the maintenance of NSCs at early and late embryonic stages, respectively (Fig. 1A). Furthermore, given that Klf15-knockdown cells only retained gliogenic activities and mostly lacked neurogenic activities, it was suggested that Klf15 is essential for maintenance of NSCs with neurogenic activities during late developmental stages and thereby plays a critical role in the transition from neurogenesis to gliogenesis (Fig. 1A).

[2]. If the control of this developmental timing is disturbed, the size and shape of the brain and its cellular composition will be severely affected. In Drosophila, it has been well established that neuroblasts alter their characteristics and sequentially express stage-specific transcription factors (Hunchback → Krüppel → Pdm → Castor) and that progeny marked by this specific gene expression differentiate into neurons with distinct functions (Isshiki et al., 2001) [9]. However, such stage-specific gene expression in NSCs has not yet been clearly demonstrated in mammalian neocortical development. We previously reported that members of the Hes gene family of basic helix-loop-helix (bHLH) transcriptional repressors (Hes1/3/5) are expressed in NSCs and maintain them by inhibiting neuronal differentiation as downstream effectors of Notch signaling (Ishibashi et al., 1994; Ohtsuka et al., 1999; Kageyama and Ohtsuka, 1999; Ohtsuka et al., 2001; Hatakeyama et al., 2004) [10–14]. To shed light on the temporal alterations in characteristics of NSCs, we utilized the Hes1 promoter and generated pHes1-d2EGFP transgenic (Tg) mice in which embryonic NSCs are labelled with enhanced green fluorescent protein (EGFP) and can be isolated by fluorescence-activated cell sorting (FACS) (Ohtsuka et al., 2006) [15]. We sorted GFP+ cells from the dorsolateral telencephalon (neocortical region) of pHes1-d2EGFP Tg embryos at different developmental stages and carried out DNA microarray (GeneChip)-based gene expression profiling (Ohtsuka et al., 2011) [3] (Fig. 1A). Among dozens of transcription factors differentially expressed in NSCs at different embryonic stages, it was revealed by in situ hybridization that several genes exhibited distinct temporal expression patterns in the ventricular zone (VZ) of neocortical regions (Fig. 1B). Based on these results, we proposed that embryonic NSCs can be categorized into at least four stages by a combination of expression patterns of multiple transcription factors, including Jarid2 (jumonji, AT rich interactive domain 2), Tcf7l1 (transcription factor 7 like 1; Tcf3), Trp53 (transformation related protein 53), Tcf4 (transcription factor 4), Klf15 (Krüppel-like factor 15), and Csdc2 (cold shock domain containing C2; Pippin) (Fig. 1C). Okamoto et al. also performed gene expression profiling of neocortical NSCs at different developmental stages by single-cell transcriptome analysis and reported differentially expressed genes during the course of neocortical development. Intriguingly, they demonstrated that temporal alterations in gene expression and identity of NSCs proceeded independently of cell cycle progression (Okamoto et al., 2016) [16]. Next, we carried out gain-of-function and loss-of-function analyses by overexpression or knockdown of those candidate genes. When we performed in utero electroporation at E13.5 and analyzed three days later at E16.5 (Fig. 1D), Tcf7l1 and Klf15 exhibited the activity to inhibit neuronal differentiation and maintain NSCs in the neocortical regions. Taking the temporal expression patterns of each gene into consideration, it was suggested that Tcf7l1 and Klf15 play critical roles in the maintenance of NSCs during early and late embryonic stages, respectively (Fig. 1A). Next, we isolated GFP+ transfected cells at E14.5 following in utero electroporation at E13.5 and carried out neurosphere forming assays using Tcf7l1- or Klf15-overexpressing cells and Tcf7l1or Klf15-knockdown cells (Fig. 1D). We found that Tcf7l1- or Klf15overexpressing cells generated significantly more primary neurospheres compared to the control, whereas the number of neurospheres generated from Tcf7l1- or Klf15-knockdown cells was less than that of control, indicating that Tcf7l1 and Klf15 are essential for the maintenance/ proliferation of NSCs. Furthermore, we cultured those primary neurospheres in differentiation conditions for five days and examined the cell lineages immunocytochemically with TUJ1 as a neuronal marker, GFAP as an astrocyte marker, and GalC as an oligodendrocyte marker (Fig. 1E). Intriguingly, whereas nearly all spheres generated astrocytes (A), neurons (N), and oligodendrocytes (O) (A/N/O) in other cases, most spheres originating from Klf15-knockdown cells generated only glial cells with a lack of differentiated neurons (A/O), indicating that knockdown of Klf15 led to the loss of neurogenic sphere-forming cells (Fig. 1F).

3. Gradual elongation of cell cycle length in NSCs during neocortical development In early developmental stages, a sheet of NSCs (neuroepithelial cells) vigorously expands through repeated symmetric divisions and the neural tube distends like a balloon with a thin wall. Following several cycles of expansion of stem cell pool, NSCs in the neocortical regions convert from symmetric proliferative to asymmetric neurogenic division and begin to produce neurons (Takahashi et al., 1995, 1999) [17,18]. This transition timing is crucial in determining onset of neurogenesis and size of initial stem cell pool. However, the precise mechanisms that regulate the switching from symmetric to asymmetric division as well as the onset of neurogenesis have not been fully elucidated. It has been demonstrated that the length of overall cell cycle (Tc) in NSCs gradually elongates over the course of neocortical development and approximately doubles over the period between embryonic day (E) 11 and E15. This is mostly due to elongation of the length of G1 phase (TG1) of the cell cycle (Miyama et al., 1997) [19]. It has been reported that this elongation of TG1 is accompanied by downregulation of Ccne1 (cyclin E) and Cdkn1a (p21) along with upregulation of Cdkn1b (p27), Cdk2, and Ccnb1 (cyclin B) (Delalle et al., 1999; Caviness et al., 2003) [20,21], and the forced reduction in TG1 by manipulating Ccnd1 (cyclin D1) expression led to an expansion of NSCs in the developing and adult brain (Lange et al., 2009; Pilaz et al., 2009; Artegiani et al., 2011) [22–24]. One possible mechanism that regulates the timing of transition from symmetric proliferative to asymmetric neurogenic division is that the gradual elongation of cell cycle length in NSCs during neocortical development allows fate determinants that promote neuronal differentiation to accumulate to a threshold level that initiates asymmetric neurogenic division and thus acts as a biological clock regulator (Calegari and Huttner, 2003; Calegari et al., 2005; Götz and Huttner, 2005; Dehay and Kennedy, 2007) [25–28]. Thus, shorter cell cycles in NSCs will inhibit these determinants from reaching the threshold needed to initiate neuronal differentiation. An alternative possibility is that the number of cell divisions intrinsically functions as a biological clock that determines competence and marks developmental steps in NSCs. If this is the case, rapid proliferation of NSCs with shorter cell cycles will result in a precocious transition from symmetric to asymmetric division and an early onset of neurogenesis. 4. Regulation of cell cycle length by Hbp1/Ccnd1 We found that expression of high mobility group box transcription factor 1 (Hbp1) was upregulated during neurogenic stages around E13.5 through E15.5 by previous gene expression profiling of embryonic NSCs in the neocortical regions (Ohtsuka et al., 2011) [3]. Therefore, we hypothesized that Hbp1 may be an important regulator of neurogenesis and analyzed its molecular function in the neocortical development. Previous studies demonstrated that Hbp1 acts as a transcriptional repressor and functions as an inhibitor of cell cycle progression by repressing downstream targets of Wnt signaling and cell cycle-related genes, such as Ccnd1 (cyclin D1), Jun (c-jun), Mycn (N-myc), and Cdkn1a (p21) (Gartel et al., 1998; Sampson et al., 2001; Kim et al., 2006; Elfert 2

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Fig. 1. Temporal alteration of neural stem cell (NSC) properties during neocortical development. (A) Illustrative diagram summarizing gene expression profiling during neocortical development. Tcf7l1 and Klf15 inhibit neuronal differentiation from NSCs and Klf15 maintains NSCs with neurogenic activities. (B) Schematic diagram of dynamic gene expressions in each category based on in situ hybridization results. Expression levels of each gene are represented by the color depth. (C) Characterization of embryonic NSCs of each developmental stage corresponding to E11.5, E13.5, E15.5, and E17.5 by a combination of expression patterns for multiple transcription factor genes. (D) Schema of in utero electroporation and neurosphere assay. Dorsolateral part of the telencephalon (neocortical region), including the electroporated region, was excised from embryos at E14.5 following in utero electroporation at E13.5, and cells were dissociated. GFP+ transfected cells were isolated using FACS and neurosphere forming assays were carried out using Tcf7l1- or Klf15-overexpressing (pEF-Tcf7l1 or pEF-Klf15) cells and Tcf7l1- or Klf15knockdown (siTcf7l1 or siKlf15) cells. The numbers of primary neurospheres, that were grown substantially by proliferation of NSCs, were counted after seven days. Then, primary neurospheres were cultured in differentiation conditions for five days and analyzed with various neuronal and glial markers. (E) Neurosphere differentiation assay. Cell clusters derived from each sphere were immunostained with anti-TUJ1 (neurons) and anti-GFAP (astrocytes) antibodies. DAPI, nuclear staining. (F) Evaluation of cellular compositions of colonies derived from each sphere. A/N/O, colonies with astrocytes (A), neurons (N), and oligodendrocytes (O); A/O, colonies with only glial cells (astrocytes and oligodendrocytes). Adapted from Ref. [Ohtsuka et al., 2011].

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Fig. 2. Regulation of the timing of neuronal differentiation during neocortical development by controlling cell cycle progression. (A) Estimation of cell cycle length in NSCs (Pax6+) in the neocortical regions by the BrdU/EdU double labeling method. Tamoxifen (Tam) was administered at E9.5. Tc, the length of overall cell cycle; Ts, the length of S phase; NC, negative control; icKO, induced conditional knockout. n = 3; error bars, s.e.m. **p < 0.01, ***p < 0.001 (Student’s t-test). (B) A diagram illustrating the relationship between gradual elongation of cell cycle length and timing of transition from symmetric proliferative to asymmetric neurogenic division based on our observations. The hypothetical threshold for onset of asymmetric neurogenic division is indicated by a dotted line. (C) Real-time RT-PCR for Hbp1 using total RNAs prepared from NSCs in the neocortical regions of pHes1-d2EGFP transgenic mice. β-actin was used as an internal control and the values were normalized to that of the E11.5 sample. n = 3; error bars, s.e.m. (D) Real-time RT-PCR using total RNAs prepared from NSCs in the neocortical regions of pHes1-d2EGFP transgenic mice showing the temporal dynamics of Ccnd1 expression. β-actin was used as an internal control and the values were normalized to that of the E11.5 sample. n = 3; error bars, s.e.m. Adapted from Ref. [Watanabe et al., 2015].

5. Regulation of transition timing of neurogenesis/gliogenesis by epigenetic factors

et al., 2013; Yan et al., 2014) [29–33]. We generated Hbp1 induced conditional knockout (icKO) mice by crossing with Nestin-CreERT2 mice and found that onset of neuronal differentiation was delayed in the neocortical regions of Hbp1 icKO mice and that NSCs continued to proliferate with a shorter cell cycle length (Watanabe et al., 2015) [34]. We analyzed the cell cycle length in Pax6+ NSCs and confirmed the gradual elongation of Tc over the course of neocortical development in both control (NC) and mutant (Hbp1 icKO) neocortices (Fig. 2A). Furthermore, we found that both Tc and the length of S phase (Ts) were significantly shorter in NSCs in the neocortical regions of Hbp1 icKO mice than in the control. Although Ts was also shortened in the mutant neocortex, it was less prominent than the reduction in Tc and was relatively constant during this period. These results indicate that the duration of the G2-M-G1 phase was significantly shortened in neocortical NSCs of Hbp1 icKO mice. Furthermore, we revealed that downstream target genes of Wnt signaling, such as Ccnd1 and Jun, were upregulated in the germinal zone of the neocortical regions of Hbp1 icKO mice. Real-time RT-PCR using total RNAs prepared from NSCs in the neocortical regions of pHes1-d2EGFP Tg mice revealed that the temporal dynamics of Ccnd1 expression exhibited a striking contrast to that of Hbp1 expression (Fig. 2C,D). We further performed a reporter assay using a Ccnd1 promoter (3.3 kb)-luciferase construct and found that Hbp1 significantly repressed the Ccnd1 promoter activity (Watanabe et al., 2015) [34]. These results suggest that Hbp1 elongates the cell cycle length through modulating Ccnd1 expression levels. Our findings supported the hypothesis that the transition timing from symmetric proliferative to asymmetric neurogenic division is determined not by the number of cell divisions but by the cell cycle length in NSCs, because rapidly proliferating NSCs in the neocortex of Hbp1 icKO mice were maintained as NSCs and the onset of neurogenesis was delayed. These results suggest that Hbp1 plays a critical role in regulating the timing of neocortical neurogenesis by elongating the cell cycle length to facilitate the threshold length required to commence neurogenesis, as illustrated in Fig. 2B.

It has been reported that temporal modification of chromatin structure by the polycomb group (PcG) complex of transcriptional repressors is one of the key mechanisms regulating the developmental timing of neocortical neurogenesis and gliogenesis (Vogel et al., 2006; Hirabayashi et al., 2009; Schwartz and Pirrotta, 2013; Pereira et al., 2010; Morimoto-Suzki et al., 2014; Corley and Kroll, 2015) [35–40]. There are two classes of polycomb repressive complex (PRC); PRC1 contains ubiquitin ligase Ring1A/B and PcG RING finger (PCGF) proteins, and PRC2 is composed of Eed, Suz12, and methyltransferase Ezh1/2 (Schwartz and Pirrotta, 2013) [37]. It is known that Ring1B functions to terminate the generation of Ctip2+ neurons by suppressing Fezf2, a fate determinant of Ctip2+ subcerebral projection neurons (SCPNs; a class of layer V neurons), suggesting that Ring1B regulates the switching from deep to superficial layer neurogenesis by timed termination of Fezf2 expression (Morimoto-Suzki et al., 2014) [39]. In addition, it was reported that levels of histone H3 lysine 27 trimethylation (H3K27me3) at the Neurog1 promoter region gradually increase over time and that PcG proteins suppress the Neurog1 locus during the gliogenic period, thereby regulating the neurogenic-to-gliogenic fate switching in the developing neocortex (Hirabayashi et al., 2009) [36]. Furthermore, it is known that the high mobility group AT-hook (Hmga) genes regulate gene expression by modulating chromatin structure (Ozturk et al., 2014) [41], maintain neurogenic NSCs, and inhibit gliogenesis during early- to mid-embryonic stages through global opening of the chromatin state (Kishi et al., 2012) [42]. Previous studies have revealed the critical roles of Hmga genes in self-renewal of NSCs (Nishino et al., 2008) [43] and gliogenesis in brain development (Sanosaka et al., 2008) [44]. It was revealed that Hmga2 promotes selfrenewal of NSCs by decreasing p16Ink4a/p19Arf expression and that Hmga2 expression declines with age, partly due to the increasing expression of let-7b microRNA (Nishino et al., 2008) [43]. In addition, it has been reported that Hmga factors are essential for the open chromatin state in early-stage NSCs to maintain neurogenic potential of 4

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Fig. 3. Regulation of transition timing of neocortical neurogenesis/gliogenesis by Hes5 and Hmga1/2. (A) Structures of pNestin-rtTA and TRE-Hes5/d2EGFP transgenes. (B) Birth date analysis of cortical neurons in wild type (WT) and Hes5-overexpressing transgenic (Tg) mice. EdU was administered at E13.5 and then localization and fate of EdU-incorporated cells (red) were examined by the layer-specific markers Ctip2 (layer V neurons) and Cux1 (layer II-IV neurons) (green) at E18.5. (C) Graphs showing the proportion of each layer-specific neurons among EdU+ cells. Total numbers of EdU+ cells counted for quantification were ≥100 cells per analysis. (D) Analysis of generation of astrocytes in the neocortical regions of WT and Tg brains at E17.5 and P0. Double staining was performed using anti-GFAP (astrocytes) (red) and anti-GFP (green) antibodies in coronal sections. (E) Real-time RT-PCR for GFAP expression using total RNAs prepared from the neocortical regions of WT and Tg brains at E16.5 and E18.5. Gapdh was used as an internal control. (F) In situ hybridization of Hmga1 and Hmga2 mRNA expression in coronal sections of WT versus Hes5-overexpressing Tg brains (left panels) and WT versus Hes5 KO brains (right panels) at E13.5. Note that color development time was shorter for the data in the left panels than in the right panels. (G) GeneChip analysis data showing upregulation of Hes5 and downregulation of Hmga1/2 in NSCs during the course of neocortical development. (H) Diagram illustrating the time scale of neurogenesis and gliogenesis in Hes5-overexpressing Tg mice, wild type, and Hes5 KO mice during neocortical development. Tbr1, marker of layer VI neurons. (B,D,F) Scale bars: 200 μm. (C,E) Data represent the mean ± s.e.m. (n = 3); *p < 0.05 (Student’s t-test). Adapted from Ref. [Bansod et al., 2017].

timing of neurogenesis and gliogenesis through altering expression levels of Hmga genes. In fact, results of GeneChip analyses exhibited a gradual upregulation of Hes5, especially between E11.5 and E13.5, and a gradual downregulation of Hmga1/2 in NSCs during the course of neocortical development (Fig. 3G). It is known that proneural genes such as Neurog2 and Ascl1 cooperatively regulate the transition from neurogenesis to gliogenesis (Nieto et al., 2001) [47]. Dennis et al. recently reported that Neurog2 and Ascl1 control the timing of neuronal laminar fate transitions by promoting deep layer neurogenesis through influencing other temporal regulators and the derepression circuit (Dennis et al., 2017) [48]. In addition to these cell-intrinsic mechanisms, transition timing of neurogenesis can be influenced by extrinsic cues. Toma et al. demonstrated that the transition from deep to superficial layer neurogenesis is regulated by negative feedback from deep layer neurons (Toma et al., 2011) [49].

NSCs during early developmental stages and that overexpression of Hmga recovers neurogenic potential even in late-stage NSCs, while knockdown of Hmga1/2 promoted gliogenesis (Kishi et al., 2012) [42]. Fujii et al. reported that insulin-like growth factor 2 mRNA-binding protein 2 (Igf2bp2) is one of the key mediators of Hmga function and regulates neurogenic potential of early-stage NSCs (Fujii et al., 2013) [45]. Despite these findings, the mechanism by which the expression of these epigenetic factors is regulated remains unclear and the mutual regulatory mechanism between the PcG complex and Hmga factors remains to be elucidated. 6. Regulation of transition timing of neurogenesis/gliogenesis by Hes5/Hmga As we mentioned in Section 2, members of the Hes gene family (Hes1/3/5) inhibit neuronal differentiation and maintain NSCs as downstream effectors of Notch signaling. After the transition from symmetric proliferative to asymmetric neurogenic division, differentiating neurons express Notch ligands such as Delta-like (Dll) and Jagged (Jag), and trigger a boost of Notch-Hes signaling in NSCs. Hes5 expression is upregulated in RGCs after the onset of neurogenesis, when NSCs start to receive strong intercellular Delta-Notch signals from differentiating and mature neurons. Thus, Hes5 is a key regulator of the maintenance of NSCs after the transition to the asymmetric neurogenic division mode. To unravel the diverse functions of Hes5 throughout the course of neocortical development, we generated Tg mouse lines in which Hes5 expression in NSCs can be manipulated by the Tet-On system (Fig. 3A) (Bansod et al., 2017) [46]. As expected, neuronal differentiation from NSCs was strongly inhibited in the neocortical regions of Hes5-overexpressing Tg mice; however, the transition from deep to superficial layer neurogenesis shifted earlier (Fig. 3B,C) and gliogenesis was accelerated and enhanced (Fig. 3D,E). In contrast, transition timing of neurogenesis and gliogenesis was delayed in the Hes5 KO mice. These alterations in transition timing of neurogenesis/gliogenesis in the neocortical regions of Hes5-overexpressing Tg, wild-type (WT), and Hes5 KO mice are summarized in Fig. 3H. We found that expression of Hmga1/2 was suppressed in the neocortical regions of Hes5-overexpressing Tg mice and that expression was conversely upregulated in the Hes5 KO brain (Fig. 3F). These results suggest that downregulation of Hmga1/2 accelerated the transition from deep to superficial layer neurogenesis as well as the onset of gliogenesis in the Hes5-overexpressing Tg neocortex. Given that Fezf2 expression was downregulated in the neocortical regions of Tg mice (Bansod et al., 2017) [46], it is likely that this accelerated transition is partly due to higher activity of PcG complexes and that reduction of Fezf2 led to earlier termination of generation of Ctip2+ deep layer neurons with precocious switching to production of Cux1+ superficial layer neurons (Fig. 3B,C). Furthermore, we found that Hes5 expression led to suppression of the Hmga1/2 promoter activity in reporter assays (Bansod et al., 2017) [46]. These results suggest that Hes5 regulates the

7. Perspectives Through gene expression profiling of embryonic NSCs using pHes1d2EGFP Tg mice, we aimed to find stage-specific markers that sharply demarcate each developmental stage. However, we could not find such genes with prominent expression in the VZ at specific time points. Therefore, it is still difficult to clearly distinguish NSCs of each developmental stage by single transcription factors, unlike the sequential expression of transcription factors in Drosophila neuroblasts. Although we focused on transcription factors, genes in other categories can possibly act as stage-specific markers during neocortical development. If such stage-specific markers are uncovered in the future, they would be very useful tools in identifying stage-specific NSCs during neocortical development and even in the process of inducing desirable neurons from embryonic stem (ES) cells or induced pluripotent stem (iPS) cells for cell replacement therapy. Our analyses of the Hbp1 icKO mice supported the hypothesis that gradual elongation of cell cycle length in NSCs during neocortical development acts as a biological clock that determines the transition timing from symmetric proliferative to asymmetric neurogenic division as well as the onset of neurogenesis. This occurs by allowing determinants for neuronal differentiation to accumulate to threshold levels during the cell cycle. However, the substance of such accumulating determinants has not yet been fully elucidated. Given that Hes factors such as Hes1 and Hes5 exhibit oscillatory expression in NSCs by a negative autoregulation mechanism (Shimojo et al., 2008; Imayoshi et al., 2013) [50,51] and result in oscillatory expression of proneural genes such as Neurog2 (Shimojo et al., 2008) [50], one possible mechanism is that the oscillatory expression and function of Neurog2 gradually modify chromatin structure and facilitate expression of neurogenic target genes such as Neurod during the elongated cell cycle (Lin et al., 2017) [52]. Our results indicated that oscillatory expression of Hes5 is also important because the precise control of Hes5 expression within appropriate levels is crucial for the regulation of transition timing of 6

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neurogenesis and gliogenesis and for normal neocortical development. It has been reported that HES5 expression in the developing human brain gradually increased during the mid-gestation period, while HMGA2 expression successively decreased (Patterson et al., 2014) [53]. Intriguingly, Patterson et al. reported a link between the let-7/HMGA2 circuit and Notch signaling. They found that HMGA2 regulates fate decisions between neurogenesis and gliogenesis in human NSCs via HES5 and demonstrated that knockdown of HMGA2 downregulated HES5 expression, probably due to blocking access of intracellular domain of Notch (NICD) to the HES5 promoter. Moreover, Parry et al. recently reported that NOTCH signaling represses HMGA1 and HMGA2 in various cell lines in a cell autonomous or non-cell autonomous manner via stimulation by the NOTCH ligand JAG1 and modulates chromatin structure by both HMGA1-dependent and independent mechanisms (Parry et al., 2018) [54]. These results and our findings together implicate a mutual regulatory mechanism between Notch signaling and the epigenetic regulatory system. Further analyses will work to uncover the full picture regarding the correlation between these two systems.

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