Plećaš Ante, Kapuralin Katarina, Grandverger Leonarda, Mitrečić Dinko, Alić Ivan
Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Zagreb, Zagreb, Croatia.
Faculty of Biotechnology and Drug Development, University of Rijeka, Rijeka, Croatia.
Cell Death Discov. 2025 Jan 22;11(1):18. doi: 10.1038/s41420-025-02297-z.
The differentiation of mouse neurons is a complex process involving cell maturation and branching, occurring during both, embryonic development and differentiation in vitro. To study mouse neuronal morphology, we used the Thy1 YFP-16 mouse strain. Although this mouse strain was described over twenty years ago, detailed studies on projections outgrowth and morphology of neurons are still lacking. The main goal of our study was to analyse the differentiation patterns of neural stem cells, including markers of differentiation, colocalization patterns, synaptic markers and the tracing of cell projections during differentiation in vitro. The neural stem cells were isolated from embryos at embryonic day 14.5 as well as newborn pups and differentiated into neurons and astrocytes. Our data showed a significant decrease of neural stem cells markers and a substantial increase in neuronal markers during differentiation, analysed by immunocytochemistry, quantitative PCR and western blot. To assess synaptic maturation, neurons were further analysed by quantitative PCR and immunocytochemistry. Expression of synaptic markers were increased during differentiation in vitro. At the 7 day in vitro differentiation, expression of synaptic markers in both YFP positive and YFP negative neurons were at comparable levels. Finally, our data revealed a significant increase in all measured morphological parameters: Filament Area, Filament Length, Filament No. Terminal Points and Sholl Intersections in YFP positive/MAP2 positive neurons compared to YFP negative/MAP2 positive neurons. These findings suggest that YFP is an effective tool for cell tracing both in vivo and in vitro, making it valuable for morphological studies during development as well as in the context of neurodegenerative disorders.
小鼠神经元的分化是一个复杂的过程，涉及细胞成熟和分支，在胚胎发育和体外分化过程中都会发生。为了研究小鼠神经元形态，我们使用了Thy1 YFP-16小鼠品系。尽管这个小鼠品系在二十多年前就已被描述，但仍缺乏对神经元突起生长和形态的详细研究。我们研究的主要目标是分析神经干细胞的分化模式，包括分化标志物、共定位模式、突触标志物以及体外分化过程中细胞突起的追踪。神经干细胞从胚胎第14.5天的胚胎以及新生幼崽中分离出来，并分化为神经元和星形胶质细胞。通过免疫细胞化学、定量PCR和蛋白质印迹分析，我们的数据显示在分化过程中神经干细胞标志物显著减少，神经元标志物大幅增加。为了评估突触成熟度，通过定量PCR和免疫细胞化学对神经元进行了进一步分析。体外分化过程中突触标志物的表达增加。在体外分化第7天，YFP阳性和YFP阴性神经元中突触标志物的表达水平相当。最后，我们的数据显示，与YFP阴性/MAP2阳性神经元相比，YFP阳性/MAP2阳性神经元中所有测量的形态学参数均显著增加：丝状面积、丝长度、丝的终端点数和Sholl交点数。这些发现表明，YFP是体内和体外细胞追踪的有效工具，对发育过程以及神经退行性疾病背景下的形态学研究具有重要价值。
