Hbp1 Regulates the Timing of Neuronal Differentiation During Cortical
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© 2015. Published by The Company of Biologists Ltd | Development (2015) 142, 2278-2290 doi:10.1242/dev.120477 RESEARCH ARTICLE STEM CELLS AND REGENERATION Hbp1 regulates the timing of neuronal differentiation during cortical development by controlling cell cycle progression Naoki Watanabe1,2,3, Ryoichiro Kageyama1,2,3,4,5 and Toshiyuki Ohtsuka1,2,3,4,* ABSTRACT number of NSCs and the size of ventricles, which largely In the developing mammalian brain, neural stem cells (NSCs) determines the eventual size and morphology of the brain. initially expand the progenitor pool by symmetric divisions. NSCs However, the precise mechanisms that regulate the switch from then shift from symmetric to asymmetric division and commence symmetric to asymmetric division and the onset of neurogenesis neurogenesis. Although the precise mechanisms regulating the have not been fully elucidated. developmental timing of this transition have not been fully It has been reported that the length of the G1 phase of the cell elucidated, gradual elongation in the length of the cell cycle and cycle (TG1) in neural stem/progenitor cells gradually rises over the coinciding accumulation of determinants that promote neuronal course of cortical development. This elongation of TG1 is – differentiation might function as a biological clock that regulates the accompanied by downregulation of cyclin E (Ccne1 Mouse – onset of asymmetric division and neurogenesis. We conducted gene Genome Informatics) and p21 (Cdkn1a Mouse Genome – expression profiling of embryonic NSCs in the cortical regions and Informatics), along with upregulation of p27 (Cdkn1b Mouse found that expression of high mobility group box transcription factor Genome Informatics), cdk2 and cyclin B (Delalle et al., 1999; 1(Hbp1) was upregulated during neurogenic stages. Induced Caviness et al., 2003). Moreover, the forced reduction of TG1 by conditional knockout mice of Hbp1, generated by crossing with manipulation of cyclin D1 led to an expansion of neural progenitor Nestin-CreERT2 mice, exhibited a remarkable dilatation of the cells in the developing and adult brain (Lange et al., 2009; Pilaz telencephalic vesicles with a tangentially expanded ventricular et al., 2009; Artegiani et al., 2011). One proposed mechanism for the zone and a thinner cortical plate containing reduced numbers of transition from symmetric proliferative to asymmetric neurogenic neurons. In these Hbp1-deficient mouse embryos, neural stem/ division is that an elongation of TG1 allows fate determinants progenitor cells continued to divide with a shorter cell cycle length. that promote neuronal differentiation to accumulate during the G1 Moreover, downstream target genes of the Wnt signaling, such as phase and exert their neurogenic functions (Calegari and Huttner, cyclin D1 (Ccnd1) and c-jun (Jun), were upregulated in the germinal 2003; Calegari et al., 2005; Götz and Huttner, 2005; Dehay and zone of the cortical regions. These results indicate that Hbp1 plays a Kennedy, 2007). crucial role in regulating the timing of cortical neurogenesis by To identify temporal alterations in the transcriptional properties of elongating the cell cycle and that it is essential for normal cortical embryonic NSCs, we previously carried out DNA microarray-based development. gene expression profiling of embryonic NSCs prepared from the cortical regions at different developmental stages by using pHes1- KEY WORDS: Hbp1, Brain morphology, Cell cycle, Cortical d2EGFP transgenic mice, which express enhanced green fluorescent development, Neural stem cells, Neuronal differentiation, Mouse protein (EGFP) in NSCs (Ohtsuka et al., 2006, 2011). Among a variety of genes that were differentially expressed during the course INTRODUCTION of development, we hypothesized that high mobility group box During mammalian cortical development, neural stem cells (NSCs) transcription factor 1 (Hbp1) might be an important regulator of gradually alter their characteristics and generate a variety of cell neurogenesis, given that it was upregulated during neurogenic stages types, which establishes the functional complexity of the brain around embryonic day 13.5 (E13.5) through embryonic day 15.5 (Temple, 2001). In early developmental stages, a sheet of NSCs (E15.5). Previous studies demonstrated that Hbp1 acts as a vigorously expands through repeated symmetric divisions, and the transcriptional repressor that functions as a cell cycle inhibitor by neural tube distends like a balloon, with a thin wall composed of repressing downstream targets of the Wnt signaling and cell cycle- short neuroepithelial cells. After expanding the progenitor pool for related genes, cyclin D1 (Ccnd1), c-jun (Jun), N-myc (Mycn)andp21 several cycles, NSCs in the cortical regions begin to produce (Cdkn1a) (Gartel et al., 1998; Sampson et al., 2001; Kim et al., 2006; neurons by converting from symmetric to asymmetric division, Elfert et al., 2013; Yan et al., 2014). which initiates neurogenesis (Takahashi et al., 1995, 1999). The Here, we analyzed the molecular function of Hbp1 in neuronal timing of this transition is crucial for determining the overall differentiation during cortical development. We found that Hbp1 controls the length of the cell cycle in neural stem/progenitor cells 1 by modulating the expression levels of cyclin D1, thereby regulating Institute for Virus Research, Kyoto University, Shogoin-Kawahara, Sakyo-ku, Kyoto 606-8507, Japan. 2Japan Science and Technology Agency, Core Research for the timing of neuronal differentiation during early cortical Evolutional Science and Technology (CREST), 4-1-8 Honcho, Kawaguchi, Saitama development. 332-0012, Japan. 3Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan. 4Graduate School of Biostudies, Kyoto University, Kyoto 606-8501, Japan. 5World Premier International Research Initiative-Institute for Integrated Cell-Material RESULTS Sciences (WPI-iCeMS), Kyoto University, Kyoto 606-8501, Japan. Hbp1 is expressed in the germinal zone during cortical *Author for correspondence ([email protected]) neurogenesis By evaluating our gene expression profiling data for embryonic Received 3 December 2014; Accepted 26 May 2015 NSCs in the developing cortex (Ohtsuka et al., 2011), we selected DEVELOPMENT 2278 RESEARCH ARTICLE Development (2015) 142, 2278-2290 doi:10.1242/dev.120477 candidate genes expected to be involved in the regulation of stages and confirmed the temporal dynamics of Hbp1 expression neuronal differentiation based on their upregulation during the indicated by microarray analysis (Fig. 1C). neurogenic period. Among these genes, we observed that Hbp1 was prominently expressed in the germinal zone that comprises the Overexpression of Hbp1 suppresses cell proliferation, but ventricular zone (VZ) and subventricular zone (SVZ) in the inhibits terminal neuronal differentiation developing telencephalon (Fig. 1A). The expression pattern of We conducted overexpression experiments byin utero electroporation. Hbp1 was characterized by a lateral high-dorsal low gradient at Hbp1 expression vectors ( pEF-Hbp1) and control vectors ( pEF- E11.5 corresponding to the propagation of neurogenesis, similar to EGFP) were co-introduced into ventricular cells in the cortical regions the expression pattern of Neurog2, a proneural (neurogenic) basic of E13.5 mouse embryos, and the fates of transfected cells were helix-loop-helix (bHLH) gene (Fig. 1B). As it was difficult to subsequently examined at E14.5 and E16.5. As shown in our previous accurately estimate alterations in Hbp1 expression levels over the paper (Ohtsuka et al., 2011), cells transfected with pEF-Hbp1 mainly course of development by in situ hybridization, we performed real- remained in the SVZ and the intermediate zone (IZ) at E16.5. time RT-PCR using total RNAs prepared from NSCs in the cortical Noticeably, a majority of transfected cells differentiated into neurons regions of pHes1-d2EGFP transgenic mice at different embryonic positive for neuronal markers, such as Tuj1 (Tubb3 – Mouse Genome Informatics) and Map2. In addition, they were negative for Ki67 (Mki67 – Mouse Genome Informatics), a marker of proliferating cells, or phospho-histone H3 (pH3), a marker of dividing cells in the M phase; moreover, they stagnated in the IZ and failed to migrate into the cortical plate (CP) (Fig. 2A; supplementary material Fig. S1A-D), indicating that neuronal migration was inhibited. The proportions of transfected cells positive for Pax6, a specific marker of NSCs during this period of development, and Tbr2 (Eomes – Mouse Genome Informatics), a marker of intermediate progenitor cells (IPs), were significantly lower than in the control at E16.5. Transfected cells that incorporated BrdU administered 4 h after in utero electroporation at E13.5 exited the VZ earlier than control cells at E15.5. However, the majority of cells still remained in the IZ at E16.5 (supplementary material Fig. S1E,F), suggesting that overexpression of Hbp1 promoted initial neuronal differentiation but impaired terminal neuronal differentiation and migration. Next, we evaluated the rate of cell proliferation by administering the thymidine analog EdU intraperitoneally to the pregnant mice 30 min before sacrifice at E14.5 to mark cells in the S phase. In cells transfected with pEF-Hbp1, EdU incorporation was suppressed compared with control (Fig. 2B), indicating that overexpression of Hbp1 caused an attenuation of cell proliferation or premature cell cycle exit. We then carried out a calculation of cell cycle kinetics