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.

在哺乳动物的嗅感觉上皮中，神经元甚至在成年期也不断产生。为了研究成年神经发生的细胞和分子机制，通过单侧切除成年小鼠的嗅球（bulbeclomy）来增强嗅上皮中的神经发生，并用溴脱氧尿苷（BrdU）标记新产生的神经元。由于增强的神经发生是更突出的犁鼻器（VNO）指定的信息素感觉，而不是在主要的嗅觉上皮气味检测，VNO在这项研究中进行了深入的研究。对照侧VNO中BrdU标记细胞稀疏分布于基底层。相反，在手术侧，许多BrdU标记的细胞出现在整个基底层，这表明大多数干细胞是静止的，但可以通过球切除术刺激增殖。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和Gi两种亚型。BrdU标记实验表明，新生神经元最初是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中的两种类型神经元起源于共同的Go阳性祖细胞。为了详细分析嗅神经发生的分子机制，利用巢蛋白启动子-EGFP转基因小鼠，其中新产生的细胞可以通过GFP荧光可视化。从其标记蛋白表达发现，VNO中的GFP表达细胞是未成熟的祖细胞。剥离转基因小鼠的感觉上皮，分散细胞，并在荧光显微镜下成功回收GFP表达细胞。该系统为进一步研究嗅神经发生的分子机制奠定了基础。