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Analysis of differentiation system in cerebral GABAergic neuron and oligodendrocyte progenitors.

脑 GABA 能神经元和少突胶质细胞祖细胞分化系统分析。

基本信息

批准号：
20700291
负责人：
ESUMI Shigeyuki
金额：
$ 2.75万
依托单位：
Kumamoto University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Young Scientists (B)
财政年份：
2008
资助国家：
日本
起止时间：
2008 至 2009
项目状态：
已结题

项目摘要

GABAergic neurons and oligodendrocyte share many characters in the forebrain development. Both cell types have been reported to originate in the medial ganglionic eminence and migrate to the neocortex. Previously, it has been reported that GABAergic neuron progenitors express not only of GABAergic neuron markers but also ones of oligodendrocytes progenitors, such as NG2 and CNP (Belachew et al., 2003 ; Aguirre et al., 2004 ; Dayer, et al., 2005). Moreover, NG2/Olig2 positive glial progenitors generate both oligodendrocyte and astrocyte in the developing forebrain using NG2creBAC transgenic mouse (Zhu et al., 2008) and Olig2-CreER knock-in mouse (Ono et al., 2008). These previous data raise a possibility that some GABAergic neuron progenitors produce glial cells in the developing forebrain. To test this possibility, here we have performed immunohistochemistry, single-cell RT-PCR, single-cell microarray analysis and immunocytochemistry analyses with glial markers in GAD67-GFP positive cells from GAD67-GFP knock-in mouse brain. As a result, we found that the neuronal markers and glial markers are co-localized in the GAD67-GFP-positive cells at mRNA and protein level. To further investigate cell lineage(s) from GABAergic neuron progenitors in vivo and in vitro, we utilized GAD67-Cre knock-in mice and Z/EG reporter mice. As a result, we found that the neuronal markers and glial markers are co-localized in the GAD67-GFP-positive cells at mRNA and protein level. Finally, to investigate GAD67 lineage in vivo and in vitro, we utilized GAD67-Cre knock-in mice and Z/EG reporter mice. We observed the most of recombined GFP positive cells were GABAergic neuron, but a few cells were oligodendrocyte and astrocyte. At present, we can not completely rule out the possibility that leaky or weak expression of GAD67 gene occur in the small subset of glial progenitors during cell-type specification.

GABA能神经元和少突胶质细胞在前脑发育过程中具有许多共同特征。据报道，这两种细胞都起源于内侧神经节隆起，并迁移到新皮质。此前，已有报道称，GABA能神经元前体细胞不仅表达GABA能神经元标记物，还表达少突胶质细胞前体细胞，如NG2和CNP(Belachew等人，2003；Aguirre等人，2004；Day er等人，2005)。此外，利用NG2creBAC转基因小鼠(朱等人，2008)和寡核苷酸基因敲入小鼠(Ono等人，2008)，NG2/Opol2阳性神经胶质前体细胞在发育中的前脑同时产生少突胶质细胞和星形胶质细胞。这些先前的数据提出了一种可能性，即一些GABA能神经元前体细胞在发育中的前脑中产生神经胶质细胞。为了验证这种可能性，我们对GAD67-GFP敲入小鼠脑中的GAD67-GFP阳性细胞进行了免疫组织化学、单细胞RT-PCR、单细胞微阵列分析和免疫细胞化学分析。结果发现，在GAD67-GFP阳性细胞中，神经元标志物和胶质标志物在mRNA和蛋白水平上是共定位的。为了进一步研究体内和体外GABA能神经元祖细胞(S)的细胞谱系，我们利用GAD67-Cre基因敲除小鼠和Z/EG报告小鼠。结果发现，在GAD67-GFP阳性细胞中，神经元标志物和胶质标志物在mRNA和蛋白水平上是共定位的。最后，为了研究GAD67在体内和体外的谱系，我们利用GAD67-Cre敲入小鼠和Z/EG报告小鼠。观察到重组GFP阳性细胞以GABA能神经元为主，少数为少突胶质细胞和星形胶质细胞。目前，我们还不能完全排除GAD67基因在细胞类型指定过程中在一小部分神经胶质前体细胞中发生泄漏或弱表达的可能性。