When the axon of a motor neuron reaches a myotube, the process of differentiation starts on both sides. This process is regulated by the exchange of signals between nerve and muscle. The growth cone of the axon is transformed into a nerve terminal, while the underlying surface of the myotube becomes distinguishable from the non-synaptic area. In the embryonic muscle fibre, acetylcholine receptors (AChR) are dispersed across the whole cell surface. With the arrival of the nerve ending, AChR concentrate below the termination area and disappear almost completely from the non-synaptic membrane. The subsequent maturation of the muscle fibre also leads to an upregulation of gene expression for AChR in those cell nuclei which are located directly beneath the nerve ending. Gene expression is downregulated in cell nuclei located outside the synaptic area. This leads to a localized synthesis of AChR in neuromuscular synapses.
Vesicles containing the neurotransmitter acetylcholine (ACh) become concentrated at specific places on the presynaptic membrane. At these sites, or active zones, the vesicles fuse together with the cell membrane and release their content into the synaptic space. At the surface of the muscle fibre, just beneath the synaptic zones, folds form in which the concentration in AChR is particularly high. The basal lamina surrounding the whole muscle fibre also becomes specialized in the synaptic region. The enzyme acetylcholinesterase accumulates there. It hydrolyzes the released ACh, thus stopping the effect of the transmitter.
Each myotube initially is innervated by the terminal branches from several different axons (polyneuronal innervation). A process then ensues by which some of these terminal branches disappear. The result of this process is mononeuronal innervation: by, or shortly before, birth each muscle fibre has contact from a single nerve terminal only. The remaining terminal then undergoes further development. Although this process of elimination means the loss of some terminal branches from individual motor neurons, it does not result in the loss of the motor axon itself. An axon losing its contacts at some muscle fibres  will gain contacts at others. Not all muscles have mononeuronal innervation in the adult state. For example, fibres in ocular muscles have multiple innervation.