HDAC1 Regulates the Proliferation of Radial Glial Cells in the Developing Xenopus Tectum

March HDAC1 Regulates the Proliferation of Radial Glial Cells in the Developing Xenopus Tectum Yi Tao 0 1 2 Hangze Ruan 0 1 2 Xia Guo 0 1 2 Lixin Li 0 1 2 Wanhua Shen 0 1 2 0 1 Department of Neurosurgery, First Affiliated Hospital of Nanjing Medical University , 300 Guangzhou Road, Nanjing, Jiangsu, 210029, China , 2 Zhejiang Key Laboratory of Organ Development and Regeneration, College of Life and Environmental Sciences, Hangzhou Normal University , Hangzhou, Zhejiang , China 1 Funding: This work was supported by grants from the National Natural Science Foundation of China (No. 81171147 to LL and No. 31271176 to WS), the Open Project Program of Zhejiang Key Laboratory of Organ Development and Regeneration to YT, the Xingwei Project Key Personal Medical Research Foundation of Health Department of Jiangsu Province (No. RC201156 to LL), the Six Categories of Key Person Research Foundation of Jiangsu Province (No. 16 069 to LL), the Priority Academic Program Development of Jiangsu Higher Education Institutions (No. JX10231801 to LL), and the Science Foundation 2 Academic Editor: Robert Blum, University of Wurzburg , GERMANY In the developing central nervous system (CNS), progenitor cells differentiate into progeny to form functional neural circuits. Radial glial cells (RGs) are a transient progenitor cell type that is present during neurogenesis. It is thought that a combination of neural trophic factors, neurotransmitters and electrical activity regulates the proliferation and differentiation of RGs. However, it is less clear how epigenetic modulation changes RG proliferation. We sought to explore the effect of histone deacetylase (HDAC) activity on the proliferation of RGs in the visual optic tectum of Xenopus laevis. We found that the number of BrdU-labeled precursor cells along the ventricular layer of the tectum decrease developmentally from stage 46 to stage 49. The co-labeling of BrdU-positive cells with brain lipid-binding protein (BLBP), a radial glia marker, showed that the majority of BrdU-labeled cells along the tectal midline are RGs. BLBP-positive cells are also developmentally decreased with the maturation of the brain. Furthermore, HDAC1 expression is developmentally down-regulated in tectal cells, especially in the ventricular layer of the tectum. Pharmacological blockade of HDACs using Trichostatin A (TSA) or Valproic acid (VPA) decreased the number of BrdUpositive, BLBP-positive and co-labeling cells. Specific knockdown of HDAC1 by a morpholino (HDAC1-MO) decreased the number of BrdU- and BLBP-labeled cells and increased the acetylation level of histone H4 at lysine 12 (H4K12). The visual deprivation-induced increase in BrdU- and BLBP-positive cells was blocked by HDAC1 knockdown at stage 49 tadpoles. These data demonstrate that HDAC1 regulates radial glia cell proliferation in the developing optical tectum of Xenopus laevis. - The maturation of the central nervous system (CNS) is mainly determined by the proliferation and differentiation of progenitor cells, which are key processes related to our understanding of cell generation with respect to brain development and repair. How progenitor cells are generated and differentiate into neurons that integrate into functional neural circuits in vivo is still relatively unknown. Radial glial cells (RGs), which originate from the neural epithelium, have for Hangzhou 131 Talents to WS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. periventricular cell bodies and single elongated processes with characteristic end feet [1]. RGs were once thought to be a subset of astroglial cells, acting only as a scaffold for the migration of newly generated neurons during the development of the CNS [2]. More recent studies have revealed that radial glia are actually a form of progenitor cells in both the developing and mature brain [36], and can proliferate and differentiate into diverse cell types to construct functional neural circuits. Elucidating the mechanisms that control the proliferation of RGs in vivo would aid in our understanding of how the brain is wired and capable of self-renewal. The proliferation of progenitor cells is regulated by intrinsic gene expression [79] and external signaling, such as through neural trophic factors [10], neurotransmitters [11] and electrical activity [12]. However, the epigenetic regulation of radial glia proliferation by histone acetylation has not been extensively studied in vivo. Histone modifications such as acetylation and deacetylation are controlled by histone acetyltransferases (HATs) and histone deacetylases (HDACs), respectively [13]. Histone acetylation by HATs provides a loose and active chromatin structure that facilitates gene transcription, whereas histone deacetylation by HDACs results in a compact and inactive chromatin structure that results in gene silencing [13]. HDACs play pivotal roles in neural development [14,15], synaptic plasticity [16], and neurological disease [17]. For example, HDAC1 regulates cardiac morphogenesis [18] and affects embryonic stem (ES) cell differentiation [19]. However, how HDACs control the fate of radial glial cell proliferation during brain development remains unknown. The HDAC family consists of four classes (class I, IIa, IIb and IV), which are highly conserved from invertebrates to mammals [17]. For this study, we investigated whether neurogenesis is regulated by HDAC1 and histone acetylation in the developing optic tectum of Xenopus laevis. To test whether RGs are actively dividing progenitor cells, we performed immunohistochemistry in the optic tectum using cell markers to identify both radial glia (with BLBP) and progenitor cells (with BrdU). We found that the majority of the BrdU-labeled precursors, which mainly distributed along the ventricular layer of the tectum were also positive for the radial glia marker. Both radial glia and precursor cells are down-regulated from developmental stages 46 to 49 in the Xenopus tectum, suggesting that the proliferation of radial glia is developmentally regulated. Bath application of an HDAC inhibitor results in a decrease in the number of BrdU- and BLBP-positive cells, indicating that HDACs are involved in radial glia proliferation. Importantly, the spatiotemporal distribution of HDAC1 is similar to that of the RGs and BrdU-labeled precursor cells in the ventricular layer of the tectum. To determine whether HDAC1 is involved in regulating the rate of radial glial cell proliferation, we used a morpholino to knockdown HDAC1 expression in the Xenopus tectum. We found that the number of BrdU-positive cells was significantly decreased compared to control animals at stage 48. Visual deprivation-induced increase of radial glia proliferation was blocked by HDAC1 knockdown at stage 49 tadpoles, suggesting that HDAC1 is required for rad (...truncated)