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Time-lapse analysis and mathematical characterization elucidate novel mechanisms underlying muscle morphogenesis.

Snow CJ, Goody M, Kelly MW, Oster EC, Jones R, Khalil A, Henry CA - PLoS Genet. (2008)

Bottom Line: These early muscle defects in the absence of either lamininbeta1 or laminingamma1 contrast with later dystrophic phenotypes in lamininalpha2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development.Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon.Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.

View Article: PubMed Central - PubMed

Affiliation: School of Biology and Ecology, University of Maine, Orono, Maine, United States of America.

ABSTRACT
Skeletal muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction (MTJ). In vertebrates, a great deal is known about muscle specification as well as how somitic cells, as a cohort, generate the early myotome. However, the cellular mechanisms that generate long muscle fibers from short cells and the molecular factors that limit elongation are unknown. We show that zebrafish fast muscle fiber morphogenesis consists of three discrete phases: short precursor cells, intercalation/elongation, and boundary capture/myotube formation. In the first phase, cells exhibit randomly directed protrusive activity. The second phase, intercalation/elongation, proceeds via a two-step process: protrusion extension and filling. This repetition of protrusion extension and filling continues until both the anterior and posterior ends of the muscle fiber reach the MTJ. Finally, both ends of the muscle fiber anchor to the MTJ (boundary capture) and undergo further morphogenetic changes as they adopt the stereotypical, cylindrical shape of myotubes. We find that the basement membrane protein laminin is required for efficient elongation, proper fiber orientation, and boundary capture. These early muscle defects in the absence of either lamininbeta1 or laminingamma1 contrast with later dystrophic phenotypes in lamininalpha2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development. Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon. Taken together, our results define three phases of muscle fiber morphogenesis and show that the critical second phase of elongation proceeds by a repetitive process of protrusion extension and protrusion filling. Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.

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lamininβ1 and γ1 are Required for Normal Fast Muscle Cell Elongation.Panels A–C are confocal images and D–J are ApoTome micrographs. Panels A–C are side views, anterior left, dorsal top of 18 somite-stage embryos stained with F59 (white) to denote slow-twitch muscle and phalloidin (red) to outline fast muscle cells. Panels labeled 1 are lateral sections from a z-series and panels labeled 2 are medial sections from the same z-series. A) In WT embryos, fast-twitch muscle cells medial (A1, white arrow) to migrating slow-twitch fibers (A, green arrow) have elongated. B) Although some slow-twitch fibers do migrate in gup/lamβ1 mutant embryos, not all fast muscle precursor cells have elongated (B1 white arrowhead: short cell, B green arrow: slow-twitch muscle fiber that has migrated laterally). C) Not all fast muscle precursor cells medial to migrating slow fibers have elongated in lamγ1 morphant embryos (C1 white arrowhead: short cell, C green arrow: slow-twitch muscle fiber that has migrated laterally). Panels D–E are projected views of dextran filled cells (red) and β-catenin that outlines cells (blue). D) Elongated fibers in a WT embryo, note the organized, parallel array of fibers. E) Elongated fibers in a lamγ1-deficient embryo, white arrowhead denotes a fiber that is not parallel. F–F2) A dextran-filled cell in a lamγ1 morphant embryo extends a thin protrusion across the MTJ. White arrows denote the MTJ, white arrowhead denotes thin protrusion extending across the MTJ. F59 denotes slow muscle in blue and dextrans are red. Panel F1 is a single focal plane from a z-series, panels F and F2 are projections. Scale bars F: 50 µm, F1: 20 µm. G–H) Cells in the myotube phase are less organized in lamγ1 morphant embryos than in control embryos as shown by more randomly oriented WTMMM vector arrows. Panels numbered 3 are higher magnification views. I–J) Although differences in cellular structure are not obvious to the eye (compare I1 and J1), lamγ1 short precursor cells are less organized than control cells as shown by more randomly oriented WTMMM vector arrows. Panels numbered 3 are higher magnification views. K: The WTMMM vector angle pdfs are displayed for all stages (color coded per panel L), the isotropic fBm surfaces (black curve fluctuating around π/2), and the flat 1/2π curve that would be obtained for a purely theoretical isotropic process (flat pointed line at 1/2π). Note the stronger (higher) peaks in control embryos. L) The anisotropy factor of muscle cells in laminin-deficient embryos is significantly lower than in control embryos at all four stages of muscle morphogenesis (p<0.01). These results indicate that even though differences in organization as far back as the precursor stage are not obvious visually, they are unequivocally more disorganized than in controls when the anisotropic value is determined.
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pgen-1000219-g006: lamininβ1 and γ1 are Required for Normal Fast Muscle Cell Elongation.Panels A–C are confocal images and D–J are ApoTome micrographs. Panels A–C are side views, anterior left, dorsal top of 18 somite-stage embryos stained with F59 (white) to denote slow-twitch muscle and phalloidin (red) to outline fast muscle cells. Panels labeled 1 are lateral sections from a z-series and panels labeled 2 are medial sections from the same z-series. A) In WT embryos, fast-twitch muscle cells medial (A1, white arrow) to migrating slow-twitch fibers (A, green arrow) have elongated. B) Although some slow-twitch fibers do migrate in gup/lamβ1 mutant embryos, not all fast muscle precursor cells have elongated (B1 white arrowhead: short cell, B green arrow: slow-twitch muscle fiber that has migrated laterally). C) Not all fast muscle precursor cells medial to migrating slow fibers have elongated in lamγ1 morphant embryos (C1 white arrowhead: short cell, C green arrow: slow-twitch muscle fiber that has migrated laterally). Panels D–E are projected views of dextran filled cells (red) and β-catenin that outlines cells (blue). D) Elongated fibers in a WT embryo, note the organized, parallel array of fibers. E) Elongated fibers in a lamγ1-deficient embryo, white arrowhead denotes a fiber that is not parallel. F–F2) A dextran-filled cell in a lamγ1 morphant embryo extends a thin protrusion across the MTJ. White arrows denote the MTJ, white arrowhead denotes thin protrusion extending across the MTJ. F59 denotes slow muscle in blue and dextrans are red. Panel F1 is a single focal plane from a z-series, panels F and F2 are projections. Scale bars F: 50 µm, F1: 20 µm. G–H) Cells in the myotube phase are less organized in lamγ1 morphant embryos than in control embryos as shown by more randomly oriented WTMMM vector arrows. Panels numbered 3 are higher magnification views. I–J) Although differences in cellular structure are not obvious to the eye (compare I1 and J1), lamγ1 short precursor cells are less organized than control cells as shown by more randomly oriented WTMMM vector arrows. Panels numbered 3 are higher magnification views. K: The WTMMM vector angle pdfs are displayed for all stages (color coded per panel L), the isotropic fBm surfaces (black curve fluctuating around π/2), and the flat 1/2π curve that would be obtained for a purely theoretical isotropic process (flat pointed line at 1/2π). Note the stronger (higher) peaks in control embryos. L) The anisotropy factor of muscle cells in laminin-deficient embryos is significantly lower than in control embryos at all four stages of muscle morphogenesis (p<0.01). These results indicate that even though differences in organization as far back as the precursor stage are not obvious visually, they are unequivocally more disorganized than in controls when the anisotropic value is determined.

Mentions: We find that muscle cell elongation in lamβ1 and γ1 mutants and morphants is delayed. In zebrafish, slow-twitch fibers migrate laterally and trigger fast muscle cell elongation [28]. Thus, slow fiber location is an excellent marker for assaying fast muscle cell elongation: fast cells medial to slow fibers should be fully elongated. Although slow muscle fiber migration is disrupted in lamβ1 and γ1-deficient embryos (Figure S1), some slow fibers migrate laterally. However, fast muscle cells medial to migrating slow fibers are short in lamβ1 or lamγ1 mutants and morphants (Figure 6B, C, and data not shown, n = 6 grumpy/lamβ1 mutant embryos, 16 lamβ1 morphant embryos, 5 wi390/lamγ1 mutant embryos and 10 lamγ1 morphant embryos).


Time-lapse analysis and mathematical characterization elucidate novel mechanisms underlying muscle morphogenesis.

Snow CJ, Goody M, Kelly MW, Oster EC, Jones R, Khalil A, Henry CA - PLoS Genet. (2008)

lamininβ1 and γ1 are Required for Normal Fast Muscle Cell Elongation.Panels A–C are confocal images and D–J are ApoTome micrographs. Panels A–C are side views, anterior left, dorsal top of 18 somite-stage embryos stained with F59 (white) to denote slow-twitch muscle and phalloidin (red) to outline fast muscle cells. Panels labeled 1 are lateral sections from a z-series and panels labeled 2 are medial sections from the same z-series. A) In WT embryos, fast-twitch muscle cells medial (A1, white arrow) to migrating slow-twitch fibers (A, green arrow) have elongated. B) Although some slow-twitch fibers do migrate in gup/lamβ1 mutant embryos, not all fast muscle precursor cells have elongated (B1 white arrowhead: short cell, B green arrow: slow-twitch muscle fiber that has migrated laterally). C) Not all fast muscle precursor cells medial to migrating slow fibers have elongated in lamγ1 morphant embryos (C1 white arrowhead: short cell, C green arrow: slow-twitch muscle fiber that has migrated laterally). Panels D–E are projected views of dextran filled cells (red) and β-catenin that outlines cells (blue). D) Elongated fibers in a WT embryo, note the organized, parallel array of fibers. E) Elongated fibers in a lamγ1-deficient embryo, white arrowhead denotes a fiber that is not parallel. F–F2) A dextran-filled cell in a lamγ1 morphant embryo extends a thin protrusion across the MTJ. White arrows denote the MTJ, white arrowhead denotes thin protrusion extending across the MTJ. F59 denotes slow muscle in blue and dextrans are red. Panel F1 is a single focal plane from a z-series, panels F and F2 are projections. Scale bars F: 50 µm, F1: 20 µm. G–H) Cells in the myotube phase are less organized in lamγ1 morphant embryos than in control embryos as shown by more randomly oriented WTMMM vector arrows. Panels numbered 3 are higher magnification views. I–J) Although differences in cellular structure are not obvious to the eye (compare I1 and J1), lamγ1 short precursor cells are less organized than control cells as shown by more randomly oriented WTMMM vector arrows. Panels numbered 3 are higher magnification views. K: The WTMMM vector angle pdfs are displayed for all stages (color coded per panel L), the isotropic fBm surfaces (black curve fluctuating around π/2), and the flat 1/2π curve that would be obtained for a purely theoretical isotropic process (flat pointed line at 1/2π). Note the stronger (higher) peaks in control embryos. L) The anisotropy factor of muscle cells in laminin-deficient embryos is significantly lower than in control embryos at all four stages of muscle morphogenesis (p<0.01). These results indicate that even though differences in organization as far back as the precursor stage are not obvious visually, they are unequivocally more disorganized than in controls when the anisotropic value is determined.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543113&req=5

pgen-1000219-g006: lamininβ1 and γ1 are Required for Normal Fast Muscle Cell Elongation.Panels A–C are confocal images and D–J are ApoTome micrographs. Panels A–C are side views, anterior left, dorsal top of 18 somite-stage embryos stained with F59 (white) to denote slow-twitch muscle and phalloidin (red) to outline fast muscle cells. Panels labeled 1 are lateral sections from a z-series and panels labeled 2 are medial sections from the same z-series. A) In WT embryos, fast-twitch muscle cells medial (A1, white arrow) to migrating slow-twitch fibers (A, green arrow) have elongated. B) Although some slow-twitch fibers do migrate in gup/lamβ1 mutant embryos, not all fast muscle precursor cells have elongated (B1 white arrowhead: short cell, B green arrow: slow-twitch muscle fiber that has migrated laterally). C) Not all fast muscle precursor cells medial to migrating slow fibers have elongated in lamγ1 morphant embryos (C1 white arrowhead: short cell, C green arrow: slow-twitch muscle fiber that has migrated laterally). Panels D–E are projected views of dextran filled cells (red) and β-catenin that outlines cells (blue). D) Elongated fibers in a WT embryo, note the organized, parallel array of fibers. E) Elongated fibers in a lamγ1-deficient embryo, white arrowhead denotes a fiber that is not parallel. F–F2) A dextran-filled cell in a lamγ1 morphant embryo extends a thin protrusion across the MTJ. White arrows denote the MTJ, white arrowhead denotes thin protrusion extending across the MTJ. F59 denotes slow muscle in blue and dextrans are red. Panel F1 is a single focal plane from a z-series, panels F and F2 are projections. Scale bars F: 50 µm, F1: 20 µm. G–H) Cells in the myotube phase are less organized in lamγ1 morphant embryos than in control embryos as shown by more randomly oriented WTMMM vector arrows. Panels numbered 3 are higher magnification views. I–J) Although differences in cellular structure are not obvious to the eye (compare I1 and J1), lamγ1 short precursor cells are less organized than control cells as shown by more randomly oriented WTMMM vector arrows. Panels numbered 3 are higher magnification views. K: The WTMMM vector angle pdfs are displayed for all stages (color coded per panel L), the isotropic fBm surfaces (black curve fluctuating around π/2), and the flat 1/2π curve that would be obtained for a purely theoretical isotropic process (flat pointed line at 1/2π). Note the stronger (higher) peaks in control embryos. L) The anisotropy factor of muscle cells in laminin-deficient embryos is significantly lower than in control embryos at all four stages of muscle morphogenesis (p<0.01). These results indicate that even though differences in organization as far back as the precursor stage are not obvious visually, they are unequivocally more disorganized than in controls when the anisotropic value is determined.
Mentions: We find that muscle cell elongation in lamβ1 and γ1 mutants and morphants is delayed. In zebrafish, slow-twitch fibers migrate laterally and trigger fast muscle cell elongation [28]. Thus, slow fiber location is an excellent marker for assaying fast muscle cell elongation: fast cells medial to slow fibers should be fully elongated. Although slow muscle fiber migration is disrupted in lamβ1 and γ1-deficient embryos (Figure S1), some slow fibers migrate laterally. However, fast muscle cells medial to migrating slow fibers are short in lamβ1 or lamγ1 mutants and morphants (Figure 6B, C, and data not shown, n = 6 grumpy/lamβ1 mutant embryos, 16 lamβ1 morphant embryos, 5 wi390/lamγ1 mutant embryos and 10 lamγ1 morphant embryos).

Bottom Line: These early muscle defects in the absence of either lamininbeta1 or laminingamma1 contrast with later dystrophic phenotypes in lamininalpha2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development.Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon.Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.

View Article: PubMed Central - PubMed

Affiliation: School of Biology and Ecology, University of Maine, Orono, Maine, United States of America.

ABSTRACT
Skeletal muscle morphogenesis transforms short muscle precursor cells into long, multinucleate myotubes that anchor to tendons via the myotendinous junction (MTJ). In vertebrates, a great deal is known about muscle specification as well as how somitic cells, as a cohort, generate the early myotome. However, the cellular mechanisms that generate long muscle fibers from short cells and the molecular factors that limit elongation are unknown. We show that zebrafish fast muscle fiber morphogenesis consists of three discrete phases: short precursor cells, intercalation/elongation, and boundary capture/myotube formation. In the first phase, cells exhibit randomly directed protrusive activity. The second phase, intercalation/elongation, proceeds via a two-step process: protrusion extension and filling. This repetition of protrusion extension and filling continues until both the anterior and posterior ends of the muscle fiber reach the MTJ. Finally, both ends of the muscle fiber anchor to the MTJ (boundary capture) and undergo further morphogenetic changes as they adopt the stereotypical, cylindrical shape of myotubes. We find that the basement membrane protein laminin is required for efficient elongation, proper fiber orientation, and boundary capture. These early muscle defects in the absence of either lamininbeta1 or laminingamma1 contrast with later dystrophic phenotypes in lamininalpha2 mutant embryos, indicating discrete roles for different laminin chains during early muscle development. Surprisingly, genetic mosaic analysis suggests that boundary capture is a cell-autonomous phenomenon. Taken together, our results define three phases of muscle fiber morphogenesis and show that the critical second phase of elongation proceeds by a repetitive process of protrusion extension and protrusion filling. Furthermore, we show that laminin is a novel and critical molecular cue mediating fiber orientation and limiting muscle cell length.

Show MeSH
Related in: MedlinePlus