Rohwedel J, Kleppisch T, Pich U, Guan K, Jin S, Zuschratter W, Hopf C, Hoch W, Hescheler J, Witzemann V, Wobus A M
In Vitro Differentiation Group, IPK Gatersleben, Germany.
Exp Cell Res. 1998 Mar 15;239(2):214-25. doi: 10.1006/excr.1997.3903.
To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, alpha 1 subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the gamma- and epsilon-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR epsilon-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiation in vitro neuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by "loss of function" analysis in vitro.
为了分析神经肌肉接头的形成，将小鼠多能胚胎干细胞通过胚状体分化为骨骼肌细胞和神经元细胞。通过逆转录聚合酶链反应（RT-PCR）分析证实了编码肌细胞生成素5、肌细胞生成素、肌分化因子和肌细胞生成素6、L型钙通道α1亚基、细胞粘附分子M-钙粘着蛋白的骨骼肌特异性基因以及编码68 kDa、160 kDa和200 kDa神经丝蛋白、突触囊泡蛋白突触素、脑特异性蛋白聚糖神经聚糖和微管相关蛋白tau的神经元特异性基因在发育过程中的表达调控。此外，还发现了在神经肌肉接头处特异性表达的基因，如烟碱型乙酰胆碱受体（AChR）的γ和ε亚基以及细胞外基质蛋白S-层粘连蛋白。在以多核自发收缩肌管形成为特征的终末分化阶段，发现肌源性调节基因肌细胞生成素6和AChR ε亚基基因（两者均在成熟成年骨骼肌中特异性表达）共同表达。只有终末分化的肌管显示出烟碱型乙酰胆碱受体（AChR）的聚集以及与聚集蛋白和突触素的共定位。通过膜片钳技术在功能水平上也证实了AChR的形成。综上所述，我们的结果表明，在体外胚胎干细胞分化过程中，神经元和肌肉特异性基因以发育调控的方式表达，导致类似突触后膜的形成。因此，胚胎干细胞分化模型将有助于通过体外“功能缺失”分析来研究神经肌肉接头处的细胞相互作用。
