Muscle phenotype of six1a morphants is associated with abnormal differentiation of fast fibers. (A–C) Lateral view of embryos at 24 hpf, (left) and high magnification of boxed regions of the trunk (right). U-shaped somites in the embryos injected with UM or SM (B, C). UM (95/100 injected embryos); SM (91/100 injected embryos). (D–F) Lateral view of embryos at 42 h. Composite bright-field and polarized light (dark-field) images of trunk muscles. Original polarized light images are shown on the right. Note the reduction of muscle birefringence in the embryos injected with UM (E) and SM (F). (G–H) Confocal saggital sections of the trunk muscle at the level of anterior somites 7–9 in control embryos (left) and morphants (right). (G, G′) Immunostaining with the cocktail of mAb A4.1025 (anti-MyHC, red), ab6301 (anti-β-Catenin, green) and DAPI (nuclei, blue). Boxed region of panel G is enlarged in panel G′. Note that the diagonal array of cross-striated fast fibers (red) was not formed and regularity of cellular shapes (green) is absent in morphants. Arrowheads point to the regions with closely positioned nuclei. (H) Staining with F310 (anti-fast MyLC, red). Note the irregular shapes of cells and absence of arrayed organization in morphant fast muscle. (I) Confocal projection of superficial region of myotome after immunostaining with F59 (anti-slow MyHC, red). Note that slow fibers retained their normal position and morphology. (J) Confocal projection of medial region of myotome after immunostaining with 4D9 (anti-Engrailed, red). Scale bars, 300 μm in panels A–C, left; 70 μm in panels A–C, right; 100 μm in panels D–F; 30 μm in panel panel G; 15 μm in panel G′; 30 μm in panels H, I; 50 μm in panel J.
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