Belliveau D J, Bechberger J F, Rogers K A, Naus C C
Department of Anatomy and Cell Biology, University of Western Ontario, London, Canada.
Dev Genet. 1997;21(3):187-200. doi: 10.1002/(SICI)1520-6408(1997)21:3<187::AID-DVG2>3.0.CO;2-3.
The P19 embryonal carcinoma cell line represents a pluripotential stem cell that can differentiate along the neural or muscle cell lineage when exposed to different environments. Exposure to retinoic acid induces P19 cells to differentiate into neurons and astrocytes that express similar developmental markers as their embryonic counterparts. We examined the expression of gap junction genes during differentiation of these stem cells into neurons and astrocytes. Untreated P19 cells express at least two gap junction proteins, connexins 26 and 43. Connexin32 could not be detected in these cells. Treatment for 96 hr with 0.3 mM retinoic acid induced the P19 cells to differentiate first into neurons followed by astrocytes. Retinoic acid produced a decrease in connexin43 mRNA, protein, and functional gap junctions. Connexin26 message was not affected by retinoic acid treatment. The neurons that developed consisted of small round cell bodies extending two to three neurites and expressed MAP2. Connexin26 was detected at sites of cell-cell and cell-neurite contact within 3 days following differentiation with retinoic acid. The astrocytes were examined for production of their intermediate filament marker, glial fibrillary acidic protein (GFAP). GFAP was first detected at 8 days by Western blotting. In culture, astrocytes co-expressed GFAP and connexin43 similar to primary cultures of mouse brain astrocytes. These results suggest that differentiation of neurons and glial cells involves specific connexin expression in each cell type. The P19 cell line will provide a valuable model with which to examine the role gap junctions play during differentiation events of developing neurons and astrocytes.
P19胚胎癌细胞系代表一种多能干细胞，当暴露于不同环境时，它可以沿着神经或肌肉细胞谱系分化。视黄酸处理可诱导P19细胞分化为神经元和星形胶质细胞，这些细胞表达与胚胎对应细胞相似的发育标志物。我们检测了这些干细胞向神经元和星形胶质细胞分化过程中缝隙连接基因的表达。未处理的P19细胞至少表达两种缝隙连接蛋白，即连接蛋白26和43。在这些细胞中未检测到连接蛋白32。用0.3 mM视黄酸处理96小时可诱导P19细胞先分化为神经元，随后分化为星形胶质细胞。视黄酸使连接蛋白43的mRNA、蛋白质和功能性缝隙连接减少。连接蛋白26的信息不受视黄酸处理的影响。分化形成的神经元由小圆形细胞体组成，伸出两到三条神经突，并表达微管相关蛋白2（MAP2）。在用视黄酸分化后的3天内，在细胞-细胞和细胞-神经突接触部位检测到连接蛋白26。检测星形胶质细胞是否产生其中间丝标志物胶质纤维酸性蛋白（GFAP）。通过蛋白质免疫印迹法在第8天首次检测到GFAP。在培养中，星形胶质细胞共表达GFAP和连接蛋白43，类似于小鼠脑星形胶质细胞的原代培养。这些结果表明，神经元和神经胶质细胞的分化涉及每种细胞类型中特定连接蛋白的表达。P19细胞系将为研究缝隙连接在发育中的神经元和星形胶质细胞分化事件中所起的作用提供一个有价值的模型。
