FORMATION OF POSTSYNAPTIC-LIKE MEMBRANES DURING DIFFERENTIATION OF EMBRYONIC STEM-CELLS IN-VITRO

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies int o skeletal muscle and neuronal cells. The developmentally controlled e xpression of skeletal muscle specific genes coding for myf5, myogenin, myoD and myf6, alpha(1) subunit of the L-type calcium channel, cell a dhesion 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-a ssociated protein tau was demonstrated by RTPCR analysis. in addition, genes specifically expressed at neuromuscular junctions, the gamma- a nd epsilon-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the termin al differentiation stage characterized by the formation of multinuclea ted spontaneously contracting myotubes, the myogenic regulatory gene m yf6 and the AChR epsilon-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the t erminally differentiated myotubes showed a clustering of nicotinic ace tylcholine receptors (AChR) and a colocalization with agrin and synapt ophysin. 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, r esulting in the formation of postsynaptic-like membranes. Thus, the em bryonic stem cell differentiation model will be helpful for studying c ellular interactions at neuromuscular junctions by ''loss of function' ' analysis in vitro. (C) 1998 Academic Press.