LIM-homeobox gene Lhx8 promote the differentiation of hippocampal newborn neurons into cholinergic neurons in vitro
LIM-homeobox gene Lhx8 promote the differentiation of hippocampal newborn neurons into cholinergic neurons in vitro
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DOI：
10.1007/s11626-013-9582-8
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发表时间：
2013-02
期刊：
影响因子：
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通讯作者：
Peipei Zhu;Haoming Li;G. Jin;Meiling Tian;X. Tan;Jinhong Shi;L. Zou;J. Qin
中科院分区：
文献类型：
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作者：
Peipei Zhu;Haoming Li;G. Jin;Meiling Tian;X. Tan;Jinhong Shi;L. Zou;J. Qin
神经发生是由神经干细胞或神经祖细胞（NPC）产生新生神经元的过程。众所周知，哺乳动物大脑有能力在两个区域产生新生神经元：脑室下区和海马齿状回的颗粒下区（SGZ）。来自SGZ中海马NPC的新生神经元迁移短距离进入颗粒细胞，并在体内与局部神经元建立突触连接（斯坦菲尔德和Trice 1988;卡梅隆等人1993）。成年海马神经发生是一个多步骤的过程，受多种内在和外在因素的调控。除NPC外，星形胶质细胞、内皮细胞、血管、细胞外基质、神经递质系统、生长因子、神经营养因子和许多其他组分也会产生复杂的微环境来调节神经发生（Vicario-Abejon et al. 2000; Doetsch 2003; Warner-Schmidt and Duman 2006; Balu and Lucki 2009）。不同神经元表型的产生也受到有丝分裂后神经元的神经元祖细胞中多种细胞外和细胞内机制的控制。同源异型盒基因作为一种内在因子控制细胞的生长、分化和迁移，并调节一些组织特异性基因（威尔金森1989; Shashikant et al. 1991; Gehring 1992）。LIM同源框基因Lhx 8位于小鼠3号染色体远端，含有两个串联重复的LIM基序和一个同源结构域。Lhx 8在基底前脑胆碱能神经元的发育和维持中具有关键和特异性作用（Kitanaka et al.1998; Mori et al.2004）。Lhx 8和另一种LIM同源结构域蛋白Lhx 6调节纹状体神经元前体分化为胆碱能和GABA能纹状体中间神经元（Fragkouli等人，2009）。在我们之前的报告中，Lhx 8的过表达促进了海马NPC在体外贴壁条件下分化为胆碱能神经元（Shi等人，2012）。然而，神经元多样性的几个方面，如索马形态、神经递质表型，通常在细胞周期退出后变得明显，并受转录因子同源框家族成员的活性控制（Shirasaki和Pfaff 2002; Fragkouli et al. 2009）。成年海马神经元起源于具有放射状胶质细胞特性的1型细胞，并通过许多中间的2型和3型细胞。快速增殖并分化为3型细胞的2型细胞逐渐退出细胞周期，随后形成未成熟和成熟神经元（Steiner et al. 2006; Knoth et al. 2010; von Bohlen und Halbach 2011）。为了进一步研究Lhx 8在神经元亚型特化中的更广泛作用，在本报告中，我们在体外获得海马新生神经元，并通过慢病毒过表达Lhx 8。我们发现Lhx 8在海马新生神经元中的过表达导致胆碱能分化。这些结果表明，新的分子策略，在体外操纵新生神经元的规格身份。我们使用GV 287慢病毒表达系统（GeneChem公司，中国上海）获得慢病毒LV-Lhx 8，如制造商的说明书。简言之，使用从胚胎第16-18天分离的RNA通过RT-PCR获得Lhx 8 cDNA（NM_001012219
Neurogenesis is a process by which newborn neurons are generated from neural stem cells or neural progenitor cells (NPCs). It is well known that the mammalian brain has the capacity to produce newborn neurons in two areas: the subventricular zone and the subgranular zone (SGZ) of the hippocampal dentate gyrus. Newborn neurons from hippocampal NPCs in SGZ migrate a short distance into granule cells and establish synaptic connection with local neurons in vivo (Stanfield and Trice 1988; Cameron et al. 1993). Adult hippocampal neurogenesis is a multiple-step process and is regulated by many factors, both intrinsic and extrinsic. In addition to NPCs, astrocytes, endothelial cells, blood vessels, extracellular matrix, neurotransmitter systems, growth factors, neurotrophic factors, and many other components create a complex microenvironment to regulate neurogenesis (Vicario-Abejon et al. 2000; Doetsch 2003; Warner-Schmidt and Duman 2006; Balu and Lucki 2009). The generation of diverse neuronal phenotypes is also controlled by multiple extracellular and intracellular mechanisms operating in neuronal progenitors of postmitotic neurons. Homeobox genes as a kind of intrinsic factors govern cellular growth, differentiation, and migration and regulate some tissue-specific genes (Wilkinson 1989; Shashikant et al. 1991; Gehring 1992). LIM-homeobox gene Lhx8 localized in the distal region of mouse chromosome 3, containing two tandemly repeated LIM motifs and a homeodomain. Lhx8 has a pivotal and specific role in the development and maintenance of basal forebrain cholinergic neurons (Kitanaka et al. 1998; Mori et al. 2004). Lhx8 and another LIM homeodomain protein Lhx6 regulated striatal neuronal precursor differentiation into cholinergic and GABAergic striatal interneurons (Fragkouli et al. 2009). In our previous report, overexpression of Lhx8 promoted the differentiation of hippocampal NPCs into cholinergic neurons on adherent conditions in vitro (Shi et al. 2012). However, several aspects of neuronal diversity, such as soma morphology, neurotransmitter phenotype, often become apparent after cell cycle exit and are controlled by the activity of members of homeobox family of transcription factors (Shirasaki and Pfaff 2002; Fragkouli et al. 2009). Adult hippocampal neurons originate from type 1 cells with radial glia cells properties and through a number of intermediate type 2 and type 3 cells. Type 2 cells with rapid proliferation and differentiated into type 3 cells gradually exit from the cell cycle and subsequently form immature and mature neurons (Steiner et al. 2006; Knoth et al. 2010; von Bohlen und Halbach 2011). To further investigate the broader role of Lhx8 in neuronal subtype specification, in this report, we acquired hippocampal newborn neurons in vitro and overexpressed Lhx8 by lentivirus. We found overexpression of Lhx8 in hippocampal newborn neurons leaded to cholinergic differentiation. These results suggested new molecular strategies for manipulating the specification identity of newborn neurons in vitro. We used a GV287 lentiviral expression system (GeneChem company, Shanghai, China) to acquired lentivirus LV-Lhx8 as manufacturer’s instructions. Briefly, the full length of Lhx8 cDNA (NM_001012219) was obtained by RT-PCR using RNA isolated from the embryonic day 16–18