Uncoupling of fast- and slow-twitch muscle morphogenesis in fn morphants. A: Cartoon showing the qualitative definition of the posterior limit of defects (PLD) that was used in this study. The PLD was considered to be the anterior most somite at which segmentally reiterated somite boundaries (albeit disrupted) were observed. B: Graph depicting posterior limit of boundary defects of embryos injected with 3- or 6-ng fn1+3 MOs. Morphant myotome defects are examined at three time points: 24 (n = 11 embryos), 32 (n = 20 embryos), and 48 (n = 13 embryos) hours postfertilization (hpf). Average PLD ± standard deviation is shown. C: Cartoon depicting the region of analysis in the embryo. Because posterior somites form in 3-ng fn1+3 morphant embryos, subsequent muscle morphogenesis was analyzed only in the posterior region (> somite 8) of 32 and 48 hpf embryos. D-I: Side views, anterior left, dorsal top, of embryos stained with F59 (green) to visualize heavy chain myosin (slow-twitch muscle) and phalloidin (red) denoting filamentous actin (fast-twitch muscle). D,G: In control embryos, MTJ boundaries are observed in slow-twitch and fast-twitch muscle (D, G, yellow arrowheads). When the two domains are overlaid, it is clear that the fast-twitch domain mirrors the slow-twitch domain (D5, D6). At 48 hpf, control embryos exhibit similar muscle morphology, with clear slow- and fast-twitch myotome boundaries and the two domains line up (G5, G6). E,H: Uncoupling of MTJ boundaries in the fast-and slow-twitch muscle domains in 3-ng fn1+3 morphant embryos. Although there are gaps in the slow-twitch domain (E1, H1, yellow asterisks), MTJ boundaries are readily apparent (yellow arrowheads). Fast-twitch fibers often cross the myotome boundary denoted by slow fibers (E4, H4, white arrowheads). This uncoupling of fast-twitch and slow-twitch morphogenesis is readily apparent when the images are overlaid (E6, H6, white arrowheads). F,I: Embryos injected with 6-ng fn1+3 MOs display more severe disruptions of MTJ boundaries, fiber morphology, and uncoupling of MTJ boundaries in the fast-twitch and slow-twitch muscle domains. J: Graph showing fast-twitch fiber and slow-twitch segment perimeters through time. In control embryos, fast-twitch and slow-twitch segment domains are approximately the same size (n = 165 myotomes). In contrast, fast and slow-twitch segment domains in fn1+3 morphant embryo are more variable (n = 126 3-ng fn morphant myotomes and n = 152 6-ng fn morphant myotomes). The trend toward a larger fast-twitch domain in fn1+3 morphant embryos reflects the uncoupling of fast-twitch and slow-twitch muscle morphogenesis. K: Graph depicting ratio of fast-twitch to slow-twitch muscle domain segment perimeter through time. The ratio was obtained by dividing the average fast-twitch myotome perimeter by the average slow-twitch myotome perimeter. There is approximately a 1:1 ratio of fast to slow muscle segment perimeters in control embryos. This ratio is increased in fn1+3 morphant embryos, indicating that fast-twitch muscle domains are larger than the corresponding slow-twitch muscle domains.
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