Limited column formation in the embryonic growth plate implies divergent growth mechanisms during pre- and postnatal bone development
- Department of Molecular Genetics, Weizmann Institute of Science, Israel
- Würzburg Institute of Systems Immunology, Julius‐Maximilians‐Universität Würzburg, Germany
- Department of Biologic and Materials & Prosthodontics, University of Michigan School of Dentistry, United States
- Aix Marseille Univ, INSERM, MMG, UMR1251, Turing Center for Living Systems, France
- School of Mathematical Sciences, Queen Mary University of London, United Kingdom
Figures
Figure 1 with 13 supplements
3D imaging of clones in the embryonic growth plate reveals complex morphologies.
Chondrocyte clones in the proximal tibia and distal femur growth plates of Col2a1-CreERT2:R26R-Confetti heterozygous mice were pulsed by tamoxifen administration at E14.5 and imaged at E18.5. (A–C) …
Figure 1—figure supplement 1
Schematic of experimental workflow and segmentation.
(A) Scheme highlighting the different stages of the modified 3D MAPs pipeline. (B) For semi-automatic segmentation of clones and nuclei, the raw 3D image is masked in Microscopy Image Browser (MIB) …
Figure 1—figure supplement 2
Orthogonal viewing angles of raw imaging data.
Images from raw data of eight embryonic clones from three orthogonal viewing angles, XY, XZ, and YZ. In each image the Y axis represents the P-D bone axis. The expectation of a column is to see a …
Figure 1—video 1
Volumetric rendering of embryonic clone in the proximal tibia growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 2
Volumetric rendering of embryonic clone in the proximal tibia growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 3
Volumetric rendering of embryonic clone in the proximal tibia growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 4
Volumetric rendering of embryonic clone in the distal femur growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 5
Volumetric rendering of embryonic clone in the distal femur growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 6
Volumetric rendering of embryonic clone in the distal femur growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 7
Volumetric rendering of embryonic clone in the distal femur growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 8
Volumetric rendering of embryonic clone in the distal femur growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 9
Volumetric rendering of embryonic clone in the distal femur growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 10
Volumetric rendering of embryonic clone in the distal femur growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 1—video 11
Volumetric rendering of embryonic clone in the distal femur growth plate.
Clone surface is in magenta and nuclear surface in blue.
Figure 2 with 2 supplements
Columns are rare in the embryonic growth plate.
Clone morphology was extracted by calculating the three orthogonal axes of each clone using principal component analysis (PCA). PC1 (pink arrow). represents the long axis of the clone, PC2 (dashed …
Figure 2—figure supplement 1
Orientation maps and distribution of uniclones in embryonic growth plates.
(A) Orientation maps of uniclones from E18.5 distal femur (DF) and proximal tibia (PT) growth plates from two orthogonal viewing angles. Each line in the map represents the orientation of the long …
Figure 2—figure supplement 2
Orientation maps and distribution of multiclones in embryonic growth plates.
(A) Orientation maps of multiclones in E18.5 distal femur (DF) and proximal tibia (PT) growth plates from two orthogonal viewing angles. Each line represents the long axis of an individual clone, …
Figure 3
3D imaging of clones in the postnatal growth plate reveals diverse and complex morphologies.
3D morphology of chondrocyte clones was analyzed in the proximal tibia (PT) and distal femur (DF) growth plates of Col2a1-CreERT2:R26R-Confetti mice. Cells were pulsed by tamoxifen administration at …
Figure 4 with 1 supplement
Complex longitudinal clones function as columns in the postnatal growth plate.
Orientation maps of clones in P40 growth plates. (A, B) Clone orientation along the P-D and D-V axes of distal femur (DF) growth plates (n = 737 columns, 1129 clusters). Asterisks indicate the same …
Figure 4—figure supplement 1
Clones orientation in the postnatal growth plate.
(A) Orientation maps along the P-D and D-V axes of single clones from distal femur (DF) (n = 1866) and proximal tibia (PT) (n = 1666) growth plates at P40. Each line represents the long axis of an …
Figure 5 with 3 supplements
A column can tolerate 60% incomplete rotations.
(A, B) Stacked histograms show the proportion of doublet cells exhibiting complete rotations (i.e., elevation angle [El] of 60–90°, in blue) vs incomplete rotations (El under 60°, in red; A) and …
Figure 5—figure supplement 1
Elevation angles between postnatal doublet cells as a function of clone size.
(A–C) Quantification of elevation angles in cell doublets from P40 distal femur (DF) growth plates (n = 737 columns, 1129 clusters). (A, B) Stacked histogram shows the proportion of doublet cells …
Figure 5—figure supplement 2
Correlation analysis between cells and nuclei.
(A) Histogram showing the distribution of elevation angles between doublet pairs of cells vs nuclei within a clone is nearly identical. Cells in red. Nuclei in blue. n = 1278 clones. (B) Pearson …
Figure 5—figure supplement 3
Noise evaluation of doublet analysis.
(A, B) Histogram showing the proportion of elevation angles from 0 to 90° of nuclei per column and per cluster as a function of number of nuclei neighbors. (C, D) Histogram showing the proportion of …
Figure 6 with 1 supplement
Growth plate expansion vs elongation ratio decreases as the bone grows.
(A) Isosurface renderings of embryonic and postnatal long bones with their chondro-osseous junction (COJ) highlighted in red. Distal femur, distal and proximal fibula, proximal humerus, distal …
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Figure 6—source data 1
Distance from longitudinal origin and equivalent radius per growth plate from E17.5 - P40.
- https://cdn.elifesciences.org/articles/95289/elife-95289-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Schematic of calculating bone expansion based on micro-CT images and raw data measurements of distance from longitudinal origin and equivalent radius.
(A) Workflow describing how growth plate expansion was calculated using equivalent radius calculations of chondro-osseous junctions. (B) Raw data showing bone and elongation and bone expansion from …
Figure 7
Model for the multifunctional design of the growth plate and its relation to bone growth.
(A) During oriented cell division in the growth plate, the division plane rotation can range from 0° to 90° (brown–blue). Less than 60° rotation indicates an incomplete rotation (red), whereas …
Author response image 1
48-hour labeling shows small clones that do not appear column-like.
3D morphology of chondrocyte clones was analyzed in the proximal tibia growth plate of Col2a1CreERT2:R26R-Confetti mice. (A) Cells were pulsed by tamoxifen administration at E15.5 and traced until …
Author response image 2
PC1 vectors overlaid on clones.
Three orthogonal viewing angles (XZ, YZ, XY) of PC1 vectors marked by black arrow overlaid on four clones from embryonic (a-d) and postnatal (e-h) growth plates. a,b,e,f are clusters, whereas …
Author response image 3
Column frequency per clone size.
Frequency of columns and clusters as a function of clone size in embryonic (A,B) and postnatal growth plates (C,D). When comparing clones of the same size, columns are more frequent postnatally.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (mouse) | Col2-CreERT | Jackson Laboratories | RRID:IMSR_JAX:006774 | |
| Strain, strain background (mouse) | R26R-Confetti | Jackson Laboratories | RRID:IMSR_JAX:017492 | |
| Sequence-based reagent | Col2CreER-F | Jackson Laboratories | PCR primers (stock # 006774) | CAC TGC GGG CTC TAC TTC AT |
| Sequence-based reagent | Col2CreER-R | Jackson Laboratories | PCR primers (stock # 006774) | ACC AGC AGC ACT TTT GGA AG |
| Sequence-based reagent | Confetti-mutant Forward | Jackson Laboratories | PCR primers (stock # 017492) | GAA TTA ATT CCG GTA TAA CTT CG |
| Sequence-based reagent | Confetti-WT Forward | Jackson Laboratories | PCR primers (stock # 017492) | AAA GTC GCT CTG AGT TGT TAT |
| Sequence-based reagent | Confetti-common | Jackson Laboratories | PCR primers (stock # 017492) | CCA GAT GAC TAC CTA TCC TC |
| Chemical compound, drug | Tamoxifen | Sigma-Aldrich | T-5648 | |
| Other | Draq5 | Thermo Scientific | 62252 | 1:2000 for embryonic samples and 1:1500 for postnatal samples |
Additional files
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Supplementary file 1
Mean and std for bone elongation and expansion measurements.
- https://cdn.elifesciences.org/articles/95289/elife-95289-supp1-v3.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/95289/elife-95289-mdarchecklist1-v3.pdf
