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doi: 10.1242/10.1242/dev.00112

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Restoration of synapse formation in Musk mutant mice expressing a Musk/Trk chimeric receptor

Ruth Herbst^*,, Ekaterina Avetisova and Steven J. Burden

Molecular Neurobiology Program, Skirball Institute, NYU Medical School, NY 10016, USA
^* Present address: Brain Research Institute, University of Vienna, Spitalgasse 4, A-1090 Vienna

View larger version (32K): [in a new window]   Fig. 1. MCK-Musk and MCK-MMT transgenes are expressed and active in skeletal muscle. (A) Expression of Musk and MMT are controlled by the 5' regulatory region of the muscle creatine kinase (MCK) gene. MCK-Musk and MCK-MMT transgenes each contain a FLAG epitope-tag in the extracellular domain of Musk (Herbst and Burden, 2000). The MCK-Musk transgene encodes a fusion protein between Musk and GFP. MMT, a chimera between the extracellular and transmembrane regions of Musk and the intracellular region of TrkA, includes a substitution of 13 amino acids from the juxtamembrane (JM) of Musk with the comparable region in TrkA (Herbst and Burden, 2000). (B) Transgene expression was measured by immunoprecipitation followed by western blotting. MCK-Musk and MCK-MMT are expressed strongly in skeletal muscle; heart expression is detected in some, but not all lines (arrows). TrkA is expressed strongly in brain. (C) Transgene expression, determined by western blotting of skeletal muscle lysates, differs among the different transgenic (Tg) lines: MCK-MMT, line 5; expresses more MMT in skeletal muscle than MCK-MMT (line 29), and MCK-Musk (line 23) expresses more Musk in skeletal muscle than MCK-Musk, line 47. (D) Musk as well as MMT are tyrosine phosphorylated. Musk and MMT were immunoprecipitated from skeletal muscle with antibodies to Musk and antibodies to TrkA, respectively, and western blots were probed with antibodies to phosphotyrosine (pY) and FLAG.

View larger version (72K): [in a new window]   Fig. 2. Synapse formation is rescued in Musk mutant mice expressing MCK-MMT or MCK-Musk. (A) Motor axons terminate at AChR clusters in muscle from E18.5 Musk mutant embryos carrying MCK-MMT or MCK-Musk. In E18.5 Musk mutant embryos, motor axons fail to stop or differentiate, and AChR clusters fail to form. (B) Synapses are singly innervated in muscle from 3-week-old Musk-/-; MCK-MMT mice, similar to wild-type and Musk-/-; MCK-Musk mice. Wholemounts of diaphragm muscles were stained with antibodies to neurofilament (green) and synaptophysin (green) to label axons and presynaptic nerve terminals, respectively, and with Texas-Red -Bungarotoxin (red) to label AChRs. Scale bar: 15 µm in A; 20 µm in B.

View larger version (72K): [in a new window]   Fig. 3. Postsynaptic proteins are clustered at synaptic sites in Musk mutant mice expressing MCK-MMT (right) Synaptic proteins, including rapsyn, ErbB4, Na+ channels and utrophin, co-localize with nerve terminals in wild-type, Musk-/-; MCK-Musk and Musk-/-; MCK-MMT mice. Sections from muscles of 3-week-old mice were stained with antibodies to postsynaptic proteins and synaptophysin (Syn) or SV2 to label presynaptic nerve terminals.

View larger version (52K): [in a new window]   Fig. 4. MCK-MMT induces ectopic, postsynaptic-like specializations. (A) In Musk-/-; MCK-MMT mice, AChRs are clustered not only at synaptic sites but also at non-synaptic sites (arrows). Wholemounts of diaphragm muscles from 3-week-old mice were stained with antibodies to neurofilament (green) and synaptophysin (green) to label axons and presynaptic nerve terminals, and with Texas-Red -Bungarotoxin (red) to label AChRs. (B) Nonsynaptic AChR clusters co-localize with other `synaptic' proteins, including rapsyn, ErbB4, Na+ channels and utrophin. Sections from muscles of 3-week-old mice were stained with antibodies to postsynaptic proteins and with Texas Red -Bungarotoxin to label AChRs. In muscle from wild-type and Musk-/-; MCK-Musk mice, nerve terminal staining reliably coincides with AChR clusters. In Musk-/-; MCK-MMT mice, nerve terminal staining coincides with only a subset of AChR clusters.

View larger version (88K): [in a new window]   Fig. 5. AChR subunit genes are transcribed selectively in subsynaptic nuclei of Musk mutant mice expressing MCK-Musk and MCK-MMT. AChR and mRNAs are expressed preferentially in the central, synaptic region of muscle (arrows) in wild-type mice and uniformly in skeletal muscle of Musk-/- mice. In Musk-/-; MCK-Musk and Musk-/-; MCK-MMT mice, AChR and mRNAs are expressed preferentially in the central region of the muscle. The pattern of AChR transcription was assessed by in situ hybridization of intercostal muscle from E18.5 embryos.

View larger version (63K): [in a new window]   Fig. 6. Musk and MMT protein are concentrated at synaptic sites, despite uniform mRNA expression in Musk-/-; MCK-Musk and Musk-/-; MCK-MMT mice. (A) Musk mRNA is expressed preferentially in the central, synaptic region of muscle in wild-type mice and at uniform, low levels in muscle from Musk mutant embryos. In Musk-/-; MCK-Musk and Musk-/-; MCK-MMT embryos, Musk and MMT mRNA, respectively, are expressed uniformly in muscle. The pattern of Musk or MMT transcription was assessed by in situ hybridization of intercostal muscle from E18.5 embryos. (B) Sections from muscle of 3-week-old mice were stained with antibodies to Musk (EC, antibodies, 83033, to an extracellular epitope; IC, antibodies, 24908, to an intracellular epitope) and with Texas Red -Bungarotoxin to label AChRs. Musk and MMT proteins are concentrated at postsynaptic sites despite uniform expression of Musk and MMT RNAs throughout the muscle. IC does not recognize MMT, as MMT lacks the epitope from the C terminus of Musk.

View larger version (62K): [in a new window]   Fig. 7. Timing of MCK expression correlates with cessation of axon growth. In E14.5 and E16.5 Musk-/-; MCK-Musk and MCK-/-; MCK-MMT embryos, motor axon growth proceeds well beyond the normal termination zone, similar to Musk mutant embryos. Motor axon growth ceases and nerve terminals differentiate only after Musk, or MMT, is expressed from the MCK regulatory region, between E16.5 and E18.5. Wholemounts of diaphragm muscles from E14.5, E16.5 and E18.5 embryos were stained with antibodies to neurofilament (NF) and synaptophysin (Syn) to label axons and presynaptic nerve terminals, respectively. (B) Quantitation of axon growth shows that motor axons stop in Musk-/-; MCK-Musk embryos at E16.5, when MCK expression begins. Likewise, in MCK-/-; MCK-MMT embryos, exuberant motor axon growth reverts at E16.5, but this reversion is partial as motor axon growth at E18.5 is intermediate between that in wild-type and Musk mutant embryos.

View larger version (87K): [in a new window]   Fig. 8. The synaptic zone remains broader in Musk-/-; MCK-Musk and Musk-/-; MCK-MMT adult mice. As a consequence of delayed expression from the MCK gene, the axon termination zone is wider in MCK-/-; MCK-Musk and MCK-/-; MCK-MMT embryos than in wild-type embryos, and this broader termination zone is retained in adult mice. Wholemounts of diaphragm muscles from 3-week-old mice were stained with antibodies to neurofilament (NF) and synaptophysin (Syn) to label axons and presynaptic nerve terminals (green), and with Texas-Red -Bungarotoxin (TR--BGT) to label AChRs (red).

View larger version (55K): [in a new window]   Fig. 9. Synaptic sites are singly innervated in Musk mutant mice expressing MCK-MMT but nerve terminal arbors are simplified. Synapses are singly innervated in muscle from 3-week-old Musk-/-; MCK-MMT mice, similar to wild-type and Musk-/-; MCK-Musk mice. Nerve terminals are similarly arborized in wild-type and Musk-/-; MCK-Musk mice (A-C), but in Musk-/-; MCK-MMT mice, nerve terminal differentiation is incomplete, ranging from terminals with less complex arbors (D) to nerve terminals with strikingly simplified arbors and bouton-like endings (E,F). Wholemounts of diaphragm muscles from three 3-week-old mice were stained with antibodies to neurofilament (NF) and synaptophysin (Syn) to label axons and presynaptic nerve terminals, respectively, and with Texas-Red -Bungarotoxin (TR--BGT) to label AChRs. Scale bar: 20 µm.