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

哺乳动物的嗅感觉上皮甚至在成人期也不断产生神经元。为了研究成年小鼠神经发生的细胞和分子机制，我们通过单侧切除嗅球（bulbecloy）来增强成年小鼠嗅上皮内的神经发生，并对新生神经元进行溴脱氧尿苷（BrdU）标记。由于神经发生的增强在用于信息素感觉的犁鼻器官（VNO）中更为突出，而不是在用于气味检测的主嗅觉上皮中，因此本研究对VNO进行了深入研究。对照侧VNO中BrdU标记细胞稀疏分布于基底层。相比之下，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，但可以通过球管切除术刺激增殖。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感觉神经元分为Go阳性和Gi阳性两种亚型。BrdU标记实验显示，新生神经元首先是Go阳性的未成熟细胞，然后分化为Go阳性和Gi阳性神经元，说明VNO中两种神经元来源于共同的Go阳性祖细胞。为了详细分析嗅觉神经发生的分子机制，我们利用巢蛋白启动子- egfp转基因小鼠，用GFP荧光观察新生成的细胞。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。剥离转基因小鼠的感觉上皮，分散细胞，荧光显微镜下成功恢复表达GFP的细胞。该系统为进一步研究体外嗅觉神经发生的分子机制提供了理论依据。

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.