Neuronal control of myogenic regulatory factor accumulation in fetal muscle.

The lumbosacral spinal cords of 14.5-day gestation mice (E14.5) were ablated. The number of molecules of each of the four myogenic regulatory factor (MRF) mRNAs per nanogram of total RNA were evaluated in innervated and aneural fetal crural muscles. Accumulation of all four MRF mRNAs was affected in aneural muscle, but was never more than threefold different than in innervated muscles, considerably less than after adult denervation. The effect of the nerve varied with the MRF, the fetal age, and with the muscle (extensor digitorum longus muscle [EDL] vs. soleus muscle), with the nerve having multiple effects including down-regulation of certain MRF genes at specific periods (e.g., myoD and myogenin [E16.5-E18.5] and MRF4 in the EDL only [E18.5-E19.5]); limiting the up-regulation of certain genes, which occurred in the absence of innervation (e.g., myf-5 [E18.5-E19.5] and myogenin [E14.5-E16.5]); and even enhancing the accumulation of MRF4 mRNA (E14.5-E16.5). We hypothesize that factors other than nerve contribute to the down-regulation of myf-5 and myogenin mRNAs to adult levels. Innervation was required for the emergence of the slow, but not the fast, MRF mRNA profile at birth. MyoD, found in both the nuclear and cytoplasmic protein extracts of innervated fetal muscle, increased by approximately 5-fold in the nuclear extracts (approximately 2.5-fold in the cytoplasmic) of E19.5 aneural muscles, significantly less than the 12-fold increase found in the nuclear extract of 4-day denervated adult muscle. This increase in aneural fetal muscle was due primarily to an increased concentration of myoD in muscle lineage nuclei, rather than to the presence of additional myoD(+) muscle lineage nuclei.

Aberrant development of motor axons and neuromuscular synapses in MyoD-null mice.

Myogenic regulatory factors (MRFs), muscle-specific transcription factors, are implicated in the activity-dependent regulation of nicotinic acetylcholine receptor (AChR) subunit genes. Here we show, with immunohistochemistry, Western blotting, and electron microscopy that MyoD, a member of the MRF family, also plays a role in fetal synapse formation. In the diaphragm of 14.5 d gestation (E14.5) wild-type and MyoD-/- mice, AChR clusters (the formation of which is under a muscle intrinsic program) are confined to a centrally located endplate zone. This distribution persists in wild-type adult muscles. However, beginning at E15.5 and extending to the adult, innervated AChR clusters are distributed all over the diaphragm of MyoD-/- mice, extending as far as the insertion of the diaphragm into the ribs. In wild-type muscle, motor axons terminate on clusters adjacent to the main intramuscular nerve; in MyoD-/- muscle, axonal bundles form extensive secondary branches that terminate on the widely distributed clusters. The number of AChR clusters on adult MyoD-/- and wild-type diaphram muscles is similar. Junctional fold density is reduced at MyoD-/- endplates, and the transition from the fetal (alpha, beta, gamma, delta) to adult-type (alpha, beta, delta, epsilon) AChRs is markedly delayed. However, MyoD-/- mice assemble a complex postsynaptic apparatus that includes muscle-specific kinase (MuSK), rapsyn, erbB, and utrophin.

Immune response to full-length dystrophin delivered to Dmd muscle by a high-capacity adenoviral vector.

Adenoviral vector-mediated gene transfer to skeletal muscle is a promising potential treatment for Duchenne muscular dystrophy. However, the immunological response to viral antigens and the therapeutic protein expressed by the delivered gene could prevent effective treatment. In this study, we investigated the immune response induced by adenoviral and dystrophin antigens presented by high-capacity adenoviral vector-mediated dystrophin and beta-galactosidase delivery to skeletal muscle of a mouse model that is both dystrophin-deficient and lacZ transgenic. Direct intramuscular gene delivery of the high-capacity adenoviral vector encoding full-length murine dystrophin resulted in stable expression of recombinant dystrophin for 5 months in mice treated as neonates and for 4 weeks in mice treated as adults. We observed neutralizing antibody to adenoviral antigens only in mice treated as adults and not in mice treated as neonates. This suggested that adenoviral antigens were only presented at the time of vector administration when the neonatal immune system was not yet mature. In contrast, antibodies to dystrophin were observed both in mice treated as neonates and in mice treated as adults. The development of an anti-dystrophin antibody response in mice treated with the high-capacity adenoviral vector as neonates suggested that dystrophin antigens were presented to the immune system at a time remote from the gene delivery, when the immune system was mature. Interestingly, an antibody response against beta-galactosidase developed late in the course of mice treated with the high-capacity adenoviral vector as neonates, suggesting a loss of tolerance to beta-galactosidase, a self-antigen in these transgenic mice. Our results suggest that future human trials of dystrophin gene delivery will need to address the potential for immunity induced by ongoing segmental degeneration of partially treated muscle fibers and presentation of recombinant dystrophin antigens in the context of a Duchenne muscular dystrophy patient.