To investigate the role of myogenin in regulating acetylcholine receptor expression in adult muscle, this muscle-specific basic helix-loop-helix transcription factor was overexpressed in transgenic mice by using regulatory elements conferring strong expression confined to differentiated postmitotic muscle fibers. Many of the transgenic mice died during the first postnatal week, but those that survived into adulthood displayed normal muscle histology, gross morphology, and motor behavior. The mRNA levels of all five acetylcholine receptor subunits (alpha, beta, gamma, delta, and epsilon) were, however, elevated. Also, the level of receptor protein was increased and high levels of receptors were present throughout the extrasynaptic surface membrane of the muscle fibers. Thus, elevated levels of myogenin are apparently sufficient to induce acetylcholine supersensitivity in normally innervated muscle of adult mice. The high neonatal mortality rate of the mice overexpressing myogenin hindered the propagation of a stable line. In an attempt to increase survival, myogenin overexpressers were mated with a line of transgenic mice overexpressing Id-1, a negative regulator that interacts with the basic helix-loop-helix family of transcription factors. The Id-1 transgene apparently worked as a second site suppressor and abolished the high rate of neonatal mortality. This effect indicates that Id-1 and myogenin interact directly or indirectly in these animals. Further study indicated that myogenin overexpression had no effect on the level of endogenous myogenin mRNA, while the levels of myoD and MRF4 mRNAs were reduced. Overexpression of the negative regulator Id-1 increased the mRNA levels of all the myogenic factors. These findings are consistent with a hypothesis suggesting that myogenic factors are influenced by mechanisms that maintain cellular homeostasis.

At mammalian neuromuscular junctions (NMJs), innervation induces and maintains the metabolic stability of acetylcholine receptors (AChRs). To explore whether neural agrin may cause similar receptor stabilization, we injected neural agrin cDNA of increasing transfection efficiencies into denervated adult rat soleus (SOL) muscles. As the efficiency increased, the amount of recombinant neural agrin expressed in the muscles also increased. This agrin aggregated AChRs on muscle fibers, whose half-life increased in a dose-dependent way from 1 to 10 days. Electrical muscle stimulation enhanced the stability of AChRs with short half-lives. Therefore, neural agrin can stabilize aggregated AChRs in a concentration-and activity-dependent way. However, there was no effect of stimulation on AChRs with a long half-life (10 days). Thus, at sufficiently high concentrations, neural agrin alone can stabilize AChRs to levels characteristic of innervated NMJs.

To study gene expression in differentiated adult motoneuron subtypes, we used fluorescent dextrans for both anterograde and retrograde axonal tracing in adult rat and mouse. Application of these dyes to the cut distal and proximal ends of small extramuscular nerve branches revealed both the peripheral ramifications and the cell bodies of subsets of motoneurons. We show that the soleus muscle is innervated by two nerve branches, one of which contains gamma motor and sensory axons but no alpha motor axons. By retrograde tracing of this branch, we selectively labelled gamma motoneurons. In adult rat, the nerves innervating the soleus and extensor digitorum longus muscles contain almost exclusively axons innervating slow (type I) and fast (type 2) muscle fibres, respectively. We selectively labelled slow and fast type motoneurons by retrograde tracing of these nerves. With immunocytochemistry we show that adult motoneurons express several homeodomain genes that are associated with motoneuron differentiation during early embryonic development. Combining selective retrograde labelling with immunocytochemistry we compared the expression patterns in alpha and gamma motoneurons. The homeodomain transcription factors Islet 1 and HB9 were expressed in slow and fast alpha motoneurons and in soleus gamma motoneurons. Motoneurons in each population varied in their intensity of the immunostaining, but no factor or combination of factors was unique to any one population.