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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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The 2D WTMM Method Is Used to Quantify Cellular Structure within a Lattice, and Indicates that Cellular Organization Increases during Muscle Morphogenesis.A) Description of how the 2D WTMM formalism quantifies structure. The starting image is of elongating muscle precursor cells stained for β-catenin to outline cells. B) Short muscle precursor cells have almost all WTMMM vector arrows pointing in random directions, indicating that there is only a small departure from isotropy (isotropy means randomly structured). C) Organization increases as muscle cells begin to elongate. Note more green arrows pointing either up or down in C than B. D) Organization continues to increase during the myotube formation phase. E) Organization is readily apparent when myotubes have formed. Note that most of the green arrows are pointing either up or down indicating high levels of organization. F) Averaged Pa(A) for one particular size scale (a∼7 µm) for the myotube stage (lime green curve), the forming myotube stage (dark green curve), elongating precursor stage (blue curve), short precursor (red curve) as well as for the isotropic fBm surfaces analyzed for calibration purposes (black curve fluctuating around π/2). Also shown is the flat 1/2π curve that would be obtained for a purely theoretical isotropic process (flat pointed line at 1/2π). G) The anisotropy factor Fa was averaged over all size scales analyzed. An indicator of organized structure, it shows significantly distinct values for all stages of developing muscle cells.
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pgen-1000219-g005: The 2D WTMM Method Is Used to Quantify Cellular Structure within a Lattice, and Indicates that Cellular Organization Increases during Muscle Morphogenesis.A) Description of how the 2D WTMM formalism quantifies structure. The starting image is of elongating muscle precursor cells stained for β-catenin to outline cells. B) Short muscle precursor cells have almost all WTMMM vector arrows pointing in random directions, indicating that there is only a small departure from isotropy (isotropy means randomly structured). C) Organization increases as muscle cells begin to elongate. Note more green arrows pointing either up or down in C than B. D) Organization continues to increase during the myotube formation phase. E) Organization is readily apparent when myotubes have formed. Note that most of the green arrows are pointing either up or down indicating high levels of organization. F) Averaged Pa(A) for one particular size scale (a∼7 µm) for the myotube stage (lime green curve), the forming myotube stage (dark green curve), elongating precursor stage (blue curve), short precursor (red curve) as well as for the isotropic fBm surfaces analyzed for calibration purposes (black curve fluctuating around π/2). Also shown is the flat 1/2π curve that would be obtained for a purely theoretical isotropic process (flat pointed line at 1/2π). G) The anisotropy factor Fa was averaged over all size scales analyzed. An indicator of organized structure, it shows significantly distinct values for all stages of developing muscle cells.

Mentions: The identification of discrete, mathematically distinct phases provides a paradigm by which muscle morphogenesis in mutant embryos can be assessed. The above data also indicate that the morphology of fixed cells is not significantly different than live cells. However, although obtaining single labeled cells within a field of unlabeled cells in fixed embryos is easier than time-lapse analysis, it is not feasible in all model systems. We thus looked for a different mathematical tool to quantify cellular organization. Ideally such a tool would allow objective quantification of cellular structure with an easier experimental preparation such as staining with phalloidin to outline all cells. Therefore, we adapted and applied the 2D Wavelet-Transform Modulus Maxima (WTMM) method [34],[35]. This method can be used to quantify the amount of structure, or order, of objects that do not necessarily have a well defined boundary. We used this approach to quantify the structural organization of cellular lattices during muscle fiber elongation. The WTMM analysis filters an image with the gradient of a smoothing function (i.e. a wavelet) at a given size scale. Places within the image where the intensity variation is maximal are given by the wavelet-transform modulus maxima (i.e. the WTMM). Next, the positions of maximal intensity variation along these maxima chains are identified. These are the WTMM maxima, or WTMMM. At these nodes, the direction where the signal has the sharpest variation is calculated. An arrow that points upward has an angle of π/2 and an arrow that points down has an angle of −π/2. The anisotropy factor Fa is then calculated from the probability density function, Pa(A), of the angles A of the WTMMM vectors. Fa is defined in such a way that randomness, isotropy, has a value of Fa = 0. Any value of Fa>0 quantifies the extent of departure from isotropy. A randomly structured cell lattice has arrows pointing in all directions and a low anisotropy factor. The arrows point in all directions because the direction of maximal intensity variation is random. A more organized cell lattice will have more arrows pointing in the same direction and a stronger anisotropic signature. More arrows will point in the same direction in an ordered cell lattice because the direction of maximal intensity variation will be the same between multiple cells. Thus, this formalism objectively provides a quantitative assessment of morphological structure. A step-by-step explanatory diagram is presented in Figure 5.


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)

The 2D WTMM Method Is Used to Quantify Cellular Structure within a Lattice, and Indicates that Cellular Organization Increases during Muscle Morphogenesis.A) Description of how the 2D WTMM formalism quantifies structure. The starting image is of elongating muscle precursor cells stained for β-catenin to outline cells. B) Short muscle precursor cells have almost all WTMMM vector arrows pointing in random directions, indicating that there is only a small departure from isotropy (isotropy means randomly structured). C) Organization increases as muscle cells begin to elongate. Note more green arrows pointing either up or down in C than B. D) Organization continues to increase during the myotube formation phase. E) Organization is readily apparent when myotubes have formed. Note that most of the green arrows are pointing either up or down indicating high levels of organization. F) Averaged Pa(A) for one particular size scale (a∼7 µm) for the myotube stage (lime green curve), the forming myotube stage (dark green curve), elongating precursor stage (blue curve), short precursor (red curve) as well as for the isotropic fBm surfaces analyzed for calibration purposes (black curve fluctuating around π/2). Also shown is the flat 1/2π curve that would be obtained for a purely theoretical isotropic process (flat pointed line at 1/2π). G) The anisotropy factor Fa was averaged over all size scales analyzed. An indicator of organized structure, it shows significantly distinct values for all stages of developing muscle cells.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1000219-g005: The 2D WTMM Method Is Used to Quantify Cellular Structure within a Lattice, and Indicates that Cellular Organization Increases during Muscle Morphogenesis.A) Description of how the 2D WTMM formalism quantifies structure. The starting image is of elongating muscle precursor cells stained for β-catenin to outline cells. B) Short muscle precursor cells have almost all WTMMM vector arrows pointing in random directions, indicating that there is only a small departure from isotropy (isotropy means randomly structured). C) Organization increases as muscle cells begin to elongate. Note more green arrows pointing either up or down in C than B. D) Organization continues to increase during the myotube formation phase. E) Organization is readily apparent when myotubes have formed. Note that most of the green arrows are pointing either up or down indicating high levels of organization. F) Averaged Pa(A) for one particular size scale (a∼7 µm) for the myotube stage (lime green curve), the forming myotube stage (dark green curve), elongating precursor stage (blue curve), short precursor (red curve) as well as for the isotropic fBm surfaces analyzed for calibration purposes (black curve fluctuating around π/2). Also shown is the flat 1/2π curve that would be obtained for a purely theoretical isotropic process (flat pointed line at 1/2π). G) The anisotropy factor Fa was averaged over all size scales analyzed. An indicator of organized structure, it shows significantly distinct values for all stages of developing muscle cells.
Mentions: The identification of discrete, mathematically distinct phases provides a paradigm by which muscle morphogenesis in mutant embryos can be assessed. The above data also indicate that the morphology of fixed cells is not significantly different than live cells. However, although obtaining single labeled cells within a field of unlabeled cells in fixed embryos is easier than time-lapse analysis, it is not feasible in all model systems. We thus looked for a different mathematical tool to quantify cellular organization. Ideally such a tool would allow objective quantification of cellular structure with an easier experimental preparation such as staining with phalloidin to outline all cells. Therefore, we adapted and applied the 2D Wavelet-Transform Modulus Maxima (WTMM) method [34],[35]. This method can be used to quantify the amount of structure, or order, of objects that do not necessarily have a well defined boundary. We used this approach to quantify the structural organization of cellular lattices during muscle fiber elongation. The WTMM analysis filters an image with the gradient of a smoothing function (i.e. a wavelet) at a given size scale. Places within the image where the intensity variation is maximal are given by the wavelet-transform modulus maxima (i.e. the WTMM). Next, the positions of maximal intensity variation along these maxima chains are identified. These are the WTMM maxima, or WTMMM. At these nodes, the direction where the signal has the sharpest variation is calculated. An arrow that points upward has an angle of π/2 and an arrow that points down has an angle of −π/2. The anisotropy factor Fa is then calculated from the probability density function, Pa(A), of the angles A of the WTMMM vectors. Fa is defined in such a way that randomness, isotropy, has a value of Fa = 0. Any value of Fa>0 quantifies the extent of departure from isotropy. A randomly structured cell lattice has arrows pointing in all directions and a low anisotropy factor. The arrows point in all directions because the direction of maximal intensity variation is random. A more organized cell lattice will have more arrows pointing in the same direction and a stronger anisotropic signature. More arrows will point in the same direction in an ordered cell lattice because the direction of maximal intensity variation will be the same between multiple cells. Thus, this formalism objectively provides a quantitative assessment of morphological structure. A step-by-step explanatory diagram is presented in Figure 5.

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