Limits...
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.

Show MeSH

Related in: MedlinePlus

Convergent-extension and mitotic division cannot account for cell spreading.(a,b) Polygon analysis of convergent-extension (CE). (a) Schematic diagrams of polygon analysis. A polygon was drawn for every cell (red) by choosing three nearest neighbours (green). Change in the height (Δh) and the width (Δw) of the polygon depends on the nature of cell motion with respect to its neighbours. (b) Δh and Δw of the PZ cells during growth (each coloured line shows the changing value of a single polygon). The increase in the mean of both Δh and Δw (thick black line) over time suggests no significant CE during the observed time window (n=472 cells, Supplementary Fig. S4e). (c–g) Angle analysis of dividing cells. Several time frames of one representative dividing cell expressing GFP (red dots) are presented (c), and the trajectories of all dividing cells were mapped (d) (n=17 cells). To analyse their trajectories, a polar coordinate system was defined (e) with r and Θ as the distance and angle with respect to xz plane between two daughter cells, respectively. If a cell divides along or orthogonal to the PDA, Θ at the time of division is 90 degrees or 0, respectively. If daughter cells undergo rearrangement after orthogonal division (f), Θ should undergo significant increase over time. (g) Θ was measured for all dividing cells and a polar histogram was employed to show that all Θ were below 15 degrees at the time of division (blue), ruling out the possibility of oriented cell division along the PDA. The fact that all Θ were below 30 degrees by the end of tracking (red) further excluded the possibility of daughter cell rearrangement afterwards (g) (n=17 cells). Scale bars, (c) 10 μm, (d) 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4403347&req=5

f3: Convergent-extension and mitotic division cannot account for cell spreading.(a,b) Polygon analysis of convergent-extension (CE). (a) Schematic diagrams of polygon analysis. A polygon was drawn for every cell (red) by choosing three nearest neighbours (green). Change in the height (Δh) and the width (Δw) of the polygon depends on the nature of cell motion with respect to its neighbours. (b) Δh and Δw of the PZ cells during growth (each coloured line shows the changing value of a single polygon). The increase in the mean of both Δh and Δw (thick black line) over time suggests no significant CE during the observed time window (n=472 cells, Supplementary Fig. S4e). (c–g) Angle analysis of dividing cells. Several time frames of one representative dividing cell expressing GFP (red dots) are presented (c), and the trajectories of all dividing cells were mapped (d) (n=17 cells). To analyse their trajectories, a polar coordinate system was defined (e) with r and Θ as the distance and angle with respect to xz plane between two daughter cells, respectively. If a cell divides along or orthogonal to the PDA, Θ at the time of division is 90 degrees or 0, respectively. If daughter cells undergo rearrangement after orthogonal division (f), Θ should undergo significant increase over time. (g) Θ was measured for all dividing cells and a polar histogram was employed to show that all Θ were below 15 degrees at the time of division (blue), ruling out the possibility of oriented cell division along the PDA. The fact that all Θ were below 30 degrees by the end of tracking (red) further excluded the possibility of daughter cell rearrangement afterwards (g) (n=17 cells). Scale bars, (c) 10 μm, (d) 50 μm.

Mentions: What, then, are the cellular processes accounting for the observed anisotropic cell spreading and metacarpal elongation? Previous qualitative studies have proposed an important role of CE in controlling cartilage morphology10. CE achieves elongation by cell-cell intercalations orthogonal to the growth axis, resulting in the narrowing and lengthening of the embryonic axis of the frog embryo, for example refs 20, 21. As our cell-cell distance analyses indicate extension in all directions (Fig. 2e,f; Supplementary Fig. 5f,g), CE could only be a significant contributor during metacarpal elongation if changes in other cellular morphogenesis compensates for the convergence. To definitively test the contribution of CE, we performed a polygon analysis, in which the relative motions of all cells with respect to their immediate neighbours are tracked over time (Fig. 3a). If CE takes place, the increase in height (along the PDA) of the polygon should accompany the decrease in its width. Using this criterion, our analysis showed that only 10% of the cells underwent CE, while 87% underwent extension-extension, that is, extension in both x and y directions (Fig. 3b, Supplementary Fig. 4e).


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)

Convergent-extension and mitotic division cannot account for cell spreading.(a,b) Polygon analysis of convergent-extension (CE). (a) Schematic diagrams of polygon analysis. A polygon was drawn for every cell (red) by choosing three nearest neighbours (green). Change in the height (Δh) and the width (Δw) of the polygon depends on the nature of cell motion with respect to its neighbours. (b) Δh and Δw of the PZ cells during growth (each coloured line shows the changing value of a single polygon). The increase in the mean of both Δh and Δw (thick black line) over time suggests no significant CE during the observed time window (n=472 cells, Supplementary Fig. S4e). (c–g) Angle analysis of dividing cells. Several time frames of one representative dividing cell expressing GFP (red dots) are presented (c), and the trajectories of all dividing cells were mapped (d) (n=17 cells). To analyse their trajectories, a polar coordinate system was defined (e) with r and Θ as the distance and angle with respect to xz plane between two daughter cells, respectively. If a cell divides along or orthogonal to the PDA, Θ at the time of division is 90 degrees or 0, respectively. If daughter cells undergo rearrangement after orthogonal division (f), Θ should undergo significant increase over time. (g) Θ was measured for all dividing cells and a polar histogram was employed to show that all Θ were below 15 degrees at the time of division (blue), ruling out the possibility of oriented cell division along the PDA. The fact that all Θ were below 30 degrees by the end of tracking (red) further excluded the possibility of daughter cell rearrangement afterwards (g) (n=17 cells). Scale bars, (c) 10 μm, (d) 50 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Convergent-extension and mitotic division cannot account for cell spreading.(a,b) Polygon analysis of convergent-extension (CE). (a) Schematic diagrams of polygon analysis. A polygon was drawn for every cell (red) by choosing three nearest neighbours (green). Change in the height (Δh) and the width (Δw) of the polygon depends on the nature of cell motion with respect to its neighbours. (b) Δh and Δw of the PZ cells during growth (each coloured line shows the changing value of a single polygon). The increase in the mean of both Δh and Δw (thick black line) over time suggests no significant CE during the observed time window (n=472 cells, Supplementary Fig. S4e). (c–g) Angle analysis of dividing cells. Several time frames of one representative dividing cell expressing GFP (red dots) are presented (c), and the trajectories of all dividing cells were mapped (d) (n=17 cells). To analyse their trajectories, a polar coordinate system was defined (e) with r and Θ as the distance and angle with respect to xz plane between two daughter cells, respectively. If a cell divides along or orthogonal to the PDA, Θ at the time of division is 90 degrees or 0, respectively. If daughter cells undergo rearrangement after orthogonal division (f), Θ should undergo significant increase over time. (g) Θ was measured for all dividing cells and a polar histogram was employed to show that all Θ were below 15 degrees at the time of division (blue), ruling out the possibility of oriented cell division along the PDA. The fact that all Θ were below 30 degrees by the end of tracking (red) further excluded the possibility of daughter cell rearrangement afterwards (g) (n=17 cells). Scale bars, (c) 10 μm, (d) 50 μm.
Mentions: What, then, are the cellular processes accounting for the observed anisotropic cell spreading and metacarpal elongation? Previous qualitative studies have proposed an important role of CE in controlling cartilage morphology10. CE achieves elongation by cell-cell intercalations orthogonal to the growth axis, resulting in the narrowing and lengthening of the embryonic axis of the frog embryo, for example refs 20, 21. As our cell-cell distance analyses indicate extension in all directions (Fig. 2e,f; Supplementary Fig. 5f,g), CE could only be a significant contributor during metacarpal elongation if changes in other cellular morphogenesis compensates for the convergence. To definitively test the contribution of CE, we performed a polygon analysis, in which the relative motions of all cells with respect to their immediate neighbours are tracked over time (Fig. 3a). If CE takes place, the increase in height (along the PDA) of the polygon should accompany the decrease in its width. Using this criterion, our analysis showed that only 10% of the cells underwent CE, while 87% underwent extension-extension, that is, extension in both x and y directions (Fig. 3b, Supplementary Fig. 4e).

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