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Dynamic imaging of the growth plate cartilage reveals multiple contributors to skeletal morphogenesis.

Li Y, Trivedi V, Truong TV, Koos DS, Lansford R, Chuong CM, Warburton D, Moats RA, Fraser SE - Nat Commun (2015)

Bottom Line: The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated.Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process.We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA [2] Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California, USA [3] Developmental Biology and Regenerative Medicine Program, Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California 90027, USA.

ABSTRACT
The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

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Cells undergo collective spreading displacement during cartilage growth.(a,b) Cells in the proliferative zone (PZ) were segmented (red dots), and their net displacement vectors (blue lines in a) and trajectories (colored lines in b) were mapped, showing strong orientation of the cell displacement towards the distal end of the tissue. (c,d) Analysis of individual cell trajectories. (c) Cell displacement over time relative to its initial y position (colour-coded as shown in the top inset), showing the largest displacement along the y axis and smaller along the x and z axes. (d) Total cell displacement (t=55 h) along different axes and planes relative to their initial y positions. Cell displacements along the y axis account for most of the displacements in 3D with linear increase in magnitude according to their initial y positions (R2=0.986), as expected if the motion of the cells depends both on local changes and similar changes happening more proximally. (e,f) Analysis of cell-cell distance change. The mean of centre-to-centre distance between all possible pairs of cells at any given time was measured (as indicated in the insets), and the distributions of those means for all cells over time (colour-coded) were plotted along the x (e) and the y axes (f) on a semi-log scale (to amplify the increase in mean over time graphically). More increase in cell-cell distance along the y axis as compared with the x axis implies an anisotropic spreading behaviour of the PZ cells; n=472 cells in (c–f). Scale bars (a,b), 50 μm.
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f2: Cells undergo collective spreading displacement during cartilage growth.(a,b) Cells in the proliferative zone (PZ) were segmented (red dots), and their net displacement vectors (blue lines in a) and trajectories (colored lines in b) were mapped, showing strong orientation of the cell displacement towards the distal end of the tissue. (c,d) Analysis of individual cell trajectories. (c) Cell displacement over time relative to its initial y position (colour-coded as shown in the top inset), showing the largest displacement along the y axis and smaller along the x and z axes. (d) Total cell displacement (t=55 h) along different axes and planes relative to their initial y positions. Cell displacements along the y axis account for most of the displacements in 3D with linear increase in magnitude according to their initial y positions (R2=0.986), as expected if the motion of the cells depends both on local changes and similar changes happening more proximally. (e,f) Analysis of cell-cell distance change. The mean of centre-to-centre distance between all possible pairs of cells at any given time was measured (as indicated in the insets), and the distributions of those means for all cells over time (colour-coded) were plotted along the x (e) and the y axes (f) on a semi-log scale (to amplify the increase in mean over time graphically). More increase in cell-cell distance along the y axis as compared with the x axis implies an anisotropic spreading behaviour of the PZ cells; n=472 cells in (c–f). Scale bars (a,b), 50 μm.

Mentions: We identified the PZ cells based on their positions in the live tissue (Supplementary Fig. 1f). To quantitatively define these cell behaviours, we performed 3D spot segmentation of individual cells, so that they could be tracked over time in a 220 × 90 × 90 μm3 region (Fig. 1g,h; Fig. 2a,b; Supplementary Movies 2–4).


Dynamic imaging of the growth plate cartilage reveals multiple contributors to skeletal morphogenesis.

Li Y, Trivedi V, Truong TV, Koos DS, Lansford R, Chuong CM, Warburton D, Moats RA, Fraser SE - Nat Commun (2015)

Cells undergo collective spreading displacement during cartilage growth.(a,b) Cells in the proliferative zone (PZ) were segmented (red dots), and their net displacement vectors (blue lines in a) and trajectories (colored lines in b) were mapped, showing strong orientation of the cell displacement towards the distal end of the tissue. (c,d) Analysis of individual cell trajectories. (c) Cell displacement over time relative to its initial y position (colour-coded as shown in the top inset), showing the largest displacement along the y axis and smaller along the x and z axes. (d) Total cell displacement (t=55 h) along different axes and planes relative to their initial y positions. Cell displacements along the y axis account for most of the displacements in 3D with linear increase in magnitude according to their initial y positions (R2=0.986), as expected if the motion of the cells depends both on local changes and similar changes happening more proximally. (e,f) Analysis of cell-cell distance change. The mean of centre-to-centre distance between all possible pairs of cells at any given time was measured (as indicated in the insets), and the distributions of those means for all cells over time (colour-coded) were plotted along the x (e) and the y axes (f) on a semi-log scale (to amplify the increase in mean over time graphically). More increase in cell-cell distance along the y axis as compared with the x axis implies an anisotropic spreading behaviour of the PZ cells; n=472 cells in (c–f). Scale bars (a,b), 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4403347&req=5

f2: Cells undergo collective spreading displacement during cartilage growth.(a,b) Cells in the proliferative zone (PZ) were segmented (red dots), and their net displacement vectors (blue lines in a) and trajectories (colored lines in b) were mapped, showing strong orientation of the cell displacement towards the distal end of the tissue. (c,d) Analysis of individual cell trajectories. (c) Cell displacement over time relative to its initial y position (colour-coded as shown in the top inset), showing the largest displacement along the y axis and smaller along the x and z axes. (d) Total cell displacement (t=55 h) along different axes and planes relative to their initial y positions. Cell displacements along the y axis account for most of the displacements in 3D with linear increase in magnitude according to their initial y positions (R2=0.986), as expected if the motion of the cells depends both on local changes and similar changes happening more proximally. (e,f) Analysis of cell-cell distance change. The mean of centre-to-centre distance between all possible pairs of cells at any given time was measured (as indicated in the insets), and the distributions of those means for all cells over time (colour-coded) were plotted along the x (e) and the y axes (f) on a semi-log scale (to amplify the increase in mean over time graphically). More increase in cell-cell distance along the y axis as compared with the x axis implies an anisotropic spreading behaviour of the PZ cells; n=472 cells in (c–f). Scale bars (a,b), 50 μm.
Mentions: We identified the PZ cells based on their positions in the live tissue (Supplementary Fig. 1f). To quantitatively define these cell behaviours, we performed 3D spot segmentation of individual cells, so that they could be tracked over time in a 220 × 90 × 90 μm3 region (Fig. 1g,h; Fig. 2a,b; Supplementary Movies 2–4).

Bottom Line: The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated.Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process.We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

View Article: PubMed Central - PubMed

Affiliation: 1] Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, California 91125, USA [2] Department of Molecular and Computational Biology, University of Southern California, Los Angeles, California, USA [3] Developmental Biology and Regenerative Medicine Program, Saban Research Institute, Children's Hospital Los Angeles, Los Angeles, California 90027, USA.

ABSTRACT
The diverse morphology of vertebrate skeletal system is genetically controlled, yet the means by which cells shape the skeleton remains to be fully illuminated. Here we perform quantitative analyses of cell behaviours in the growth plate cartilage, the template for long bone formation, to gain insights into this process. Using a robust avian embryonic organ culture, we employ time-lapse two-photon laser scanning microscopy to observe proliferative cells' behaviours during cartilage growth, resulting in cellular trajectories with a spreading displacement mainly along the tissue elongation axis. We build a novel software toolkit of quantitative methods to segregate the contributions of various cellular processes to the cellular trajectories. We find that convergent-extension, mitotic cell division, and daughter cell rearrangement do not contribute significantly to the observed growth process; instead, extracellular matrix deposition and cell volume enlargement are the key contributors to embryonic cartilage elongation.

Show MeSH
Related in: MedlinePlus