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The absence of Pax7-positive progenitor cells in nfixa-MO-injected embryos determines the loss of secondary fibers. (A-F) Immunohistochemistry experiments with the Pax7 antibody that recognizes secondary fiber precursors. Pax7-positive secondary fiber progenitors at the dorsal and ventral extremes of the superficial muscle monolayer did not form in 48 hpf and 3 dpf nfixa-MO-injected embryos (B,E) in comparison with controls (A,D arrow), whereas they are recovered in rescued embryos (C,F). (G) Western blot: secondary fiber precursors were recognized by Pax7 antibody, all sarcomeric myosins MyHC were recognized by MF20 antibody. In isolated tails of nfixa-MO-injected larvae, Pax7 protein levels were higher than the control whereas they were recovered in rescued embryos at 3 dpf. All myosin protein levels were higher in nfixa-MO-injected larvae, as expected. (A-C) 48 hpf transversal sections, dorsal on top. (D-F) 3 dpf longitudinal sections, anterior towards the left. Scale bar: 100 μm.
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