title: Formation of postsynaptic-like membranes during differentiation of embryonic stem cells in vitro.
abstract: 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.
authors.affiliation: In Vitro Differentiation Group, IPK Gatersleben, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Rohwedel
authors.forename: T
authors.initials: T
authors.lastname: Kleppisch
authors.forename: U
authors.initials: U
authors.lastname: Pich
authors.forename: K
authors.initials: K
authors.lastname: Guan
authors.forename: S
authors.initials: S
authors.lastname: Jin
authors.forename: W
authors.initials: W
authors.lastname: Zuschratter
authors.forename: C
authors.initials: C
authors.lastname: Hopf
authors.forename: W
authors.initials: W
authors.lastname: Hoch
authors.forename: J
authors.initials: J
authors.lastname: Hescheler
authors.forename: V
authors.initials: V
authors.lastname: Witzemann
authors.forename: A M
authors.initials: AM
authors.lastname: Wobus
mesh_terms: D018171:Agrin / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics; D020746:Calcium Channels, L-Type; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D005786:Gene Expression Regulation; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000235:genetics; D051379:Mice; D008856:Microscopy, Fluorescence; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics; D009469:Neuromuscular Junction / Q000166:cytology*; D011978:Receptors, Nicotinic / Q000096:biosynthesis / Q000235:genetics; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D016708:Synaptophysin / Q000096:biosynthesis / Q000235:genetics
chemical_list: D018171:Agrin; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D008565:Membrane Proteins; D009124:Muscle Proteins; D009419:Nerve Tissue Proteins; D011978:Receptors, Nicotinic; D016708:Synaptophysin