Analysis of molecular mechanisms for neurogenesis in the mammalian olfactory sensory epithelium

哺乳动物嗅觉上皮神经发生的分子机制分析

• 批准号: 13680863
• 负责人: YAMAGUCHI Masahiro
• 金额: $ 2.3万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680863/
• 关键词: neurogenesis; vomeronasal organ; nestin; GFP

Neurons are continuously generated in the mammalian olfactory sensory epithelium even in the adult period. To examine the cellular and molecular mechanism of adult neurogensis, neurogenesis in the olfactory epithelium was enhanced by the unilateral removal of the olfactory bulb (bulbeclomy) in the adult mice, and the newly-generated neurons were followed by bromodeoxyuridine (BrdU) labeling. Because the enhancement of neurogenesis was more prominent in the vomeronasal organ (VNO) specified for pheromone sensation rather than in the main olfactory epithelium for odorant detection, VNO was intensively examined in this study. In the VNO of control side, BrdU labeled ceils were sparsely located at the basal layer. In contrast, in the operated side, many BrdU labeled cells appeared throughout the basal layer, which indicated that most stem cells are quiescent but can be stimulated to proliferate with bulbectomy. Within several days BrdU labeled cells migrated to superficial layers and expressed neuronal marker proteins. There are two subtypes in the sensory neurons in VNO, the Go positive and the Gi positive neurons. BrdU labeling experiments showed that new neurons were at first Go positive immature cells and then differentiated into Go positive and Gi positive neurons, indicating that two types of neurons in the VNO originate from common Go positive progenitors. To analyze the molecular mechanism of olfactory neurogenesis in detail, nestin promoter-EGFP transgenic mice were utilized, where newly-generated cells can be visualized by GFP fluorescence. GFP expressing cells in VNO were found to be immature progenitors from their marker protein expression. The sensory epithelium of the transgenic mice was stripped, the cells were dispersed, and the GFP expressing cells were successfully recovered under fluorescent microscopy. This system was considered to be useful to further examine the molecular mechanism of olfactory neurogenesis in vitro.

在哺乳动物的嗅觉上皮细胞中，甚至在成年期也不断地产生神经元。为探讨成体神经发生的细胞和分子机制，去掉成年小鼠一侧嗅球，用溴脱氧尿嘧啶核苷(BrdU)标记新生的神经元，增强嗅觉上皮的神经发生。由于感觉信息素的犁鼻器(VNO)比嗅觉气味的主要嗅觉上皮(VO)更能促进神经发生，因此本研究对VNO进行了深入的研究。对照侧VNO中，BrdU标记细胞散在分布于基底层。而在手术侧，大量BrdU标记细胞出现在基底层，表明大部分干细胞处于静止状态，但可以通过切除球部刺激其增殖。在几天内，BrdU标记的细胞迁移到浅层，并表达神经元标记蛋白。VNO的感觉神经元有GO阳性神经元和GI阳性神经元两种亚型。BrdU标记实验显示，新神经元最初是GO阳性的未成熟细胞，然后分化为GO阳性和GI阳性神经元，表明VNO中的两种神经元来自常见的GO阳性前体细胞。为了详细分析嗅神经发生的分子机制，利用Nestin启动子-EGFP转基因小鼠，通过GFP荧光观察新生细胞。从标记蛋白的表达来看，VNO中表达GFP的细胞为未成熟祖细胞。将转基因小鼠的感觉上皮剥离，细胞分散，荧光显微镜下成功回收表达GFP的细胞。该系统可用于进一步研究嗅神经发生的分子机制。