Inaguma Yutaka, Ito Hidenori, Hara Akira, Iwamoto Ikuko, Matsumoto Ayumi, Yamagata Takanori, Tabata Hidenori, Nagata Koh-Ichi
Department of Molecular Neurobiology, Institute for Developmental Research, Aichi Human Service Center, Aichi 480-0392, Japan.
Department of Tumor Pathology, Gifu University Graduate School of Medicine, Gifu 501-1194, Japan.
Neurosci Res. 2015 Mar;92:21-8. doi: 10.1016/j.neures.2014.10.017. Epub 2014 Nov 6.
Timeless was originally identified in Drosophila as an essential component of circadian cycle regulation. In mammals, the ortholog of Timeless (Tim) has also implicated in cell cycle control and embryonic development. In this study, we generated a specific antibody against Tim, and carried out expression and localization analyses of Tim during mouse brain development. In Western blotting, Tim was detected throughout the developmental stage. In immunohistochemical analyses, Tim was detected strongly in neurons in the ventricular zone/subventricular zone and moderately in cortical neurons during corticogenesis. In adult mouse brain, Tim was observed moderately in cortical neurons. Notably, Tim was enriched in the nucleus of cortical neurons from embryonic to early postnatal stages while it was distributed in the cytoplasm in the adult stage. Similar distribution change from nucleus to cytoplasm was observed in the hippocampal neurons between P0 and P30. In situ hybridization revealed that the tissue expression profile of Tim-mRNA was similar to that of the protein. In differentiated primary cultured mouse hippocampal neurons, Tim was detected in cell body, axon and dendrites. The obtained results suggest that Tim is expressed in neuronal tissues in a spatiotemporally regulated manner and involved in developmental stage-specific neuronal functions.
“永恒”最初是在果蝇中被鉴定为昼夜节律调控的一个重要组成部分。在哺乳动物中，“永恒”的直系同源物（Tim）也与细胞周期控制和胚胎发育有关。在本研究中，我们制备了一种针对Tim的特异性抗体，并对Tim在小鼠大脑发育过程中的表达和定位进行了分析。在蛋白质免疫印迹分析中，在整个发育阶段都检测到了Tim。在免疫组织化学分析中，在脑室区/脑室下区的神经元中Tim被强烈检测到，在皮质发生过程中，皮质神经元中检测到中等水平的Tim。在成年小鼠大脑中，在皮质神经元中观察到中等水平的Tim。值得注意的是，从胚胎期到出生后早期，Tim在皮质神经元的细胞核中富集，而在成年期它分布在细胞质中。在出生后0天到30天之间的海马神经元中也观察到了从细胞核到细胞质的类似分布变化。原位杂交显示Tim-mRNA的组织表达谱与蛋白质相似。在分化的原代培养小鼠海马神经元中，在细胞体、轴突和树突中都检测到了Tim。所获得的结果表明，Tim以时空调节的方式在神经组织中表达，并参与发育阶段特异性的神经元功能。
