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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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ECM expansion and cell volume enlargement account for cell spreading.(a,b) Col2a antibody staining (red) of frozen sections from the chick metacarpal expressing cytoplasmic-GFP (green). (a) Low magnification view. (b) Enlarged image of the region in the white box in (a). Inset of (b) shows sample counterstained with DAPI (blue) to reveal the locations of cell nuclei (three experiments; n=4 per experiment). (c,d) Cell segmentation for voxel analysis. (c) Maximum intensity projections of five time points in the 4D live imaging of a chick metacarpal expressing GFP; the region enclosed within the expanding white box selected for voxel analysis, based on the same four cells on the boundaries (red dots). (d) Corresponding binary images provided clear identification of voxels as either ECM (black) and cell (white) volume. (e,f) Voxel analysis. (e) Total count of the number of dark and white voxels, denoting the volume occupied by ECM and cells, respectively, shows the expansion of both ECM and cell volume. (f) Decomposition of the increase in ECM and cell length along the x and y axes, expressed as the percentage of the length at t=0 (100% denotes no change). (g,h) Results of computer simulations of cell trajectories, based on the model of tissue growth described in text and depicted schematically in Fig. 5. (g) Overlapping simulated and experimental (tracked) cell trajectories along the y axis depicted for six randomly chosen cells. Heat map in the insert depicts the errors of all simulated trajectories as a percentage of the experimental values showing that the absolute errors are always below 3%. (h) Total cell displacement length (t=55 h) along the y axis of all simulated cells are plotted against their initial y positions, displaying similar distribution pattern to the experimental ones; n=109 cells in (c–e). Scale bars, (a,b) 15 μm, (c,d) 50 μm.
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f4: ECM expansion and cell volume enlargement account for cell spreading.(a,b) Col2a antibody staining (red) of frozen sections from the chick metacarpal expressing cytoplasmic-GFP (green). (a) Low magnification view. (b) Enlarged image of the region in the white box in (a). Inset of (b) shows sample counterstained with DAPI (blue) to reveal the locations of cell nuclei (three experiments; n=4 per experiment). (c,d) Cell segmentation for voxel analysis. (c) Maximum intensity projections of five time points in the 4D live imaging of a chick metacarpal expressing GFP; the region enclosed within the expanding white box selected for voxel analysis, based on the same four cells on the boundaries (red dots). (d) Corresponding binary images provided clear identification of voxels as either ECM (black) and cell (white) volume. (e,f) Voxel analysis. (e) Total count of the number of dark and white voxels, denoting the volume occupied by ECM and cells, respectively, shows the expansion of both ECM and cell volume. (f) Decomposition of the increase in ECM and cell length along the x and y axes, expressed as the percentage of the length at t=0 (100% denotes no change). (g,h) Results of computer simulations of cell trajectories, based on the model of tissue growth described in text and depicted schematically in Fig. 5. (g) Overlapping simulated and experimental (tracked) cell trajectories along the y axis depicted for six randomly chosen cells. Heat map in the insert depicts the errors of all simulated trajectories as a percentage of the experimental values showing that the absolute errors are always below 3%. (h) Total cell displacement length (t=55 h) along the y axis of all simulated cells are plotted against their initial y positions, displaying similar distribution pattern to the experimental ones; n=109 cells in (c–e). Scale bars, (a,b) 15 μm, (c,d) 50 μm.

Mentions: Having ruled out major roles for CE, mitotic cell division and daughter cell rearrangement, we addressed the possible roles of cell volume enlargement and intercellular space expansion111213. One of the major components of the ECM filling the intercellular space in the PZ is col2a (Fig. 4a,b)12. As 97% of the PZ cells in our imaged chick metacarpals were labelled with cytoplasmic-GFP (Supplementary Fig. 2), the dark area between green cells offers a clean means to recognize the space occupied by ECM, thus allowing us to simultaneously determine the contributions of both cell and ECM volumes. We selected a region in the distal PZ (white box in Fig. 4c) and segmented the image into cell (GFP bright) and ECM (GFP dark) (Fig. 4d); the results showed a 62% increase in ECM volume and a 27% increase in cell volume (Fig. 4e). Decomposing these changes along the x and y axes revealed that both the ECM and the cells displayed anisotropic expansion, mainly along the y axis (Fig. 4f). These changes are consistent with the increase of both height and width in the earlier polygon analysis (Fig. 3a,b). Compared with 12% increase in cell size along the y axis (and 10% along the x axis), there was a 40% increase in ECM in the same direction (and 20% along x axis). Thus, the increase in the total volume of the selected region results from increases in both volumes, with ECM expansion playing the more significant role.


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)

ECM expansion and cell volume enlargement account for cell spreading.(a,b) Col2a antibody staining (red) of frozen sections from the chick metacarpal expressing cytoplasmic-GFP (green). (a) Low magnification view. (b) Enlarged image of the region in the white box in (a). Inset of (b) shows sample counterstained with DAPI (blue) to reveal the locations of cell nuclei (three experiments; n=4 per experiment). (c,d) Cell segmentation for voxel analysis. (c) Maximum intensity projections of five time points in the 4D live imaging of a chick metacarpal expressing GFP; the region enclosed within the expanding white box selected for voxel analysis, based on the same four cells on the boundaries (red dots). (d) Corresponding binary images provided clear identification of voxels as either ECM (black) and cell (white) volume. (e,f) Voxel analysis. (e) Total count of the number of dark and white voxels, denoting the volume occupied by ECM and cells, respectively, shows the expansion of both ECM and cell volume. (f) Decomposition of the increase in ECM and cell length along the x and y axes, expressed as the percentage of the length at t=0 (100% denotes no change). (g,h) Results of computer simulations of cell trajectories, based on the model of tissue growth described in text and depicted schematically in Fig. 5. (g) Overlapping simulated and experimental (tracked) cell trajectories along the y axis depicted for six randomly chosen cells. Heat map in the insert depicts the errors of all simulated trajectories as a percentage of the experimental values showing that the absolute errors are always below 3%. (h) Total cell displacement length (t=55 h) along the y axis of all simulated cells are plotted against their initial y positions, displaying similar distribution pattern to the experimental ones; n=109 cells in (c–e). Scale bars, (a,b) 15 μm, (c,d) 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: ECM expansion and cell volume enlargement account for cell spreading.(a,b) Col2a antibody staining (red) of frozen sections from the chick metacarpal expressing cytoplasmic-GFP (green). (a) Low magnification view. (b) Enlarged image of the region in the white box in (a). Inset of (b) shows sample counterstained with DAPI (blue) to reveal the locations of cell nuclei (three experiments; n=4 per experiment). (c,d) Cell segmentation for voxel analysis. (c) Maximum intensity projections of five time points in the 4D live imaging of a chick metacarpal expressing GFP; the region enclosed within the expanding white box selected for voxel analysis, based on the same four cells on the boundaries (red dots). (d) Corresponding binary images provided clear identification of voxels as either ECM (black) and cell (white) volume. (e,f) Voxel analysis. (e) Total count of the number of dark and white voxels, denoting the volume occupied by ECM and cells, respectively, shows the expansion of both ECM and cell volume. (f) Decomposition of the increase in ECM and cell length along the x and y axes, expressed as the percentage of the length at t=0 (100% denotes no change). (g,h) Results of computer simulations of cell trajectories, based on the model of tissue growth described in text and depicted schematically in Fig. 5. (g) Overlapping simulated and experimental (tracked) cell trajectories along the y axis depicted for six randomly chosen cells. Heat map in the insert depicts the errors of all simulated trajectories as a percentage of the experimental values showing that the absolute errors are always below 3%. (h) Total cell displacement length (t=55 h) along the y axis of all simulated cells are plotted against their initial y positions, displaying similar distribution pattern to the experimental ones; n=109 cells in (c–e). Scale bars, (a,b) 15 μm, (c,d) 50 μm.
Mentions: Having ruled out major roles for CE, mitotic cell division and daughter cell rearrangement, we addressed the possible roles of cell volume enlargement and intercellular space expansion111213. One of the major components of the ECM filling the intercellular space in the PZ is col2a (Fig. 4a,b)12. As 97% of the PZ cells in our imaged chick metacarpals were labelled with cytoplasmic-GFP (Supplementary Fig. 2), the dark area between green cells offers a clean means to recognize the space occupied by ECM, thus allowing us to simultaneously determine the contributions of both cell and ECM volumes. We selected a region in the distal PZ (white box in Fig. 4c) and segmented the image into cell (GFP bright) and ECM (GFP dark) (Fig. 4d); the results showed a 62% increase in ECM volume and a 27% increase in cell volume (Fig. 4e). Decomposing these changes along the x and y axes revealed that both the ECM and the cells displayed anisotropic expansion, mainly along the y axis (Fig. 4f). These changes are consistent with the increase of both height and width in the earlier polygon analysis (Fig. 3a,b). Compared with 12% increase in cell size along the y axis (and 10% along the x axis), there was a 40% increase in ECM in the same direction (and 20% along x axis). Thus, the increase in the total volume of the selected region results from increases in both volumes, with ECM expansion playing the more significant role.

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