Oriented clonal cell dynamics enables accurate growth and shaping of vertebrate cartilage
- Karolinska Institutet, Sweden
- Medical University Vienna, Austria
- Brno University of Technology, Czech Republic
- Uppsala University, Sweden
- Masaryk University, Czech Republic
- University of Michigan School of Dentistry, United States
- Technische Universität Dresden, Germany
- University of Nebraska Medical Center, United States
- Royal Institute of Technology, Sweden
- John Innes Centre, United Kingdom
- National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, United States
- Sechenov First Moscow State Medical University, Russia
Figures
Figure 1 with 2 supplements
Visualizations of endochondrial ossifications in the chondrocranium during development.
(A) 3D models of chondrocrania with visualized bone and hypertrophic cartilage. Note the absence of endochondrial ossifications in the nasal capsule between E14.5–17.5. Intramembraneous …
Figure 1—figure supplement 1
Histological confirmation of μ-CT results.
(A–F) Alcian blue (blue, stains for cartilage) and Alizarin red (red/dark brown, stains for bone) histological staining has been applied on thin sections of E17.5 (A,B), P0 (C,D) and P4 (E,F) heads. …
Figure 1—figure supplement 2
Immuno- and histological validation of cartilage contrasting obtained from µ-CT analysis and subsequent 3D modelling.
(A) Raw µ-CT scan, a sagittal view through the head of an E15.5 mouse embryo. The yellow dotted line shows the area that is presented as a corresponding 3D model in (B) and the red dotted line shows …
Figure 2 with 2 supplements
Induction of initial shape and geometrical transformations of the facial chondrocranium.
(A–C) µ-CT-based 3D reconstruction of chondrogenic mesenchymal condensations and cartilage in the face of E12.5, E13.5, E14.5 and E17.5 embryos. (A) Mesenchymal condensations (yellow) segmented from …
Figure 2—figure supplement 1
Genetic tracing serves as a tool for birth-dating of the cartilage during the embryonic development.
(A–H) Genetic tracing was induced at E12.5 in Sox10-CreERT2/R26Confetti (A–F) and Col2a1-CreERT2/R26Confetti (G, H), and all samples were analyzed at E17.5 in sagittal and transversal sections. …
Figure 2—figure supplement 2
Genetic tracing based on Ebf2-CreERT2/R26Tomato serves as an indicator for structures developed from late mesenchymal condensations.
(A–B) 3D-models of chondrocranium segmented from µ-CT scans of E14.5 (A) and E15.5 (B) embryos. Frame and magnified inset in (A) show the location where the connector structure will develop during …
Figure 3 with 2 supplements
Facial chondrocranium undergoes major lateral expansion without extensive thickening during growth.
(A) The 3D-model of E14.5 nasal capsule (blue) is placed onto the E17.5 model (green) for better presentation of growth-related changes. (B) Frontal clipping planes of 3D-models of nasal capsules at …
Figure 3—figure supplement 1
3D models and wall thickness analysis of chondrocraniums at different developmental stages.
(A) 3D models of nasal capsules created from segmentation of the raw µ-CT scans of mouse embryos at developmental stages E14.5 to E17.5. (B) Analysis of the cartilaginous wall thickness (shown by …
Figure 3—figure supplement 2
Comparisons of the shape and size differences between developmental stages and Wnt/PCP mutants.
(A) Developmental stages E13.5, E14.5, E15.5, E16.5 and E17.5 were compared to the previous developmental stage in a GOM Inspect Software. E15.5 Wnt/PCP mutants (Wnt5a-/-, Vangl2-/- and Ror2-/- …
Figure 4 with 2 supplements
Clones of chondrocytes show transversely oriented columnar structure in sheet-shaped facial cartilage.
(A–C) Chondrocyte clones at E17.5 were genetically traced from neural crest cells (E8.5), sagittal sections. The cartilage is outlined with white dashed line. (D–F) Analysis of EdU incorporation (24 …
Figure 4—figure supplement 1
Oriented clonal dynamics in chondrogenic mesenchymal condensations.
(A–C) 3D-models of chondrogenic condensations at embryonic stages 12.5 (A–B) and 13.5 (B–C). (B) E12.5 model is fitted inside of the E13.5 model for size and geometry comparisons. (C) Clipping plane …
Figure 4—figure supplement 2
Clonal oriented clusters of chondrocytes contain closely associated perichondrial cell in flat facial cartilages.
(A,B) 3D visualizations of solitary clonal columns using 150 µm-thick sections. The dotted line indicates the border of the cartilage. (C) Examples of oriented clonal clusters and columns containing …
Figure 5
Mathematical model of cell dynamics during sheet-shaped cartilage development and growth.
(A) Transversal (along z-axis) clipping plane showing conceptual arrangements of modelled cells within the layer as a result of a typical simulation. The degree of microstructure order, S, is …
Figure 6
Clonal arrangements of chondrocytes influence local geometry and cartilage surface.
(A–H) Columnar clonal arrangements in sheet-shaped cartilages of facial chondrocranium visualized with genetic tracing in Sox10-CreERT2/R26Confetti embryos. (A) 3D-model with a clipping plane shown …
Figure 7
Deficiencies in Wnt/PCP pathway reshape the chondrocranium.
(A) µ-CT-based reconstructions of the facial chondrocranium of wild type control, Wnt5a-/-, Vangl2-/-, Ror2-/- and Vangl2-/+/Ror2-/+ embryos at E15.5, with wall-thickness analysis (the row below). …
Figure 8
Manipulation of cartilage thickness in GSα mutant embryos.
(A) Wall thickness was analysed in the µ-CT segmented olfactory system of control (left) and GSα (G-protein subunit alpha) mutant (right). Large areas with decreased cartilage thickness are …
Figure 9
Oriented cell dynamics during development of rod-shaped cartilage elements.
(A) Genetic tracing in developing rib cartilage. Note the transverse pattern of chondrocyte clones. Dotted rectangle shows the area of magnified inset on bottom left. (B) 6 hr after EdU pulse in …
Figure 10
Schematic overview of cartilage shaping and scaling processes.
(1) Oriented cell divisions in the mesenchymal condensations give rise to the transverse columnar clones of chondrocytes. (2) Perichondrial cells may potentially give rise to chondrocytes. (3) …
Figure 11
Analysis of proliferation identifies specific proliferative regions in nasal capsule.
(A–B) Analysis of EdU incorporation 24 hr after the pulse on a transversal section of the facial chondrocranium at E14.5. Notice the distinct proliferative zones in the cartilage that correlate with …
Figure 12
Modeling of uneven growth in the shaping of the nasal capsule.
(A) In silico geometrical transformations of the nasal capsule-like anlage at E13.5 following various scenarios including: anisotropic oriented growth (following polarization introduced by the …
Scheme 1
Starting shapes for the Fixed mid-ridge model (A) and Non-Fixed mid-ridge model (B).
Green colour indicates the values of POL which is highest at the proximal end. CHEEKS are shown in purple and MID is shown in grey.
Appendix 1—figure 1
(A) Individual cells are modeled by a number of properties such as their color and distributions for cell division times.
The positions of cells in space are tracked on an underlying unstructured lattice, or grid. The edges in the primary mesh (solid lines) connect vertices (black dots). A biological cell is modeled by …
Appendix 1—figure 2
When a cell divides (yellow cell), the daughter cell will push surrounding cells (blue) to make room for the progeny (A).
The direction of pushing (and what cell to push) is determined by a combination of the directivity of the original division or pushing direction and of a penalty for pushing an occupied site. The …
Videos
Video 1
3D-models based on segmentation of mesenchymal condensations and mature cartilage from µ-CT tomographic data.
The first sequence illustrates wall thickness analysis results represented as a heat-map, starting from E12.5 (facial mesenchymal condensation) until E17.5 (facial cartilage). Cartilages and other …
Video 2
Simulations of shape transitions of the nasal capsule-like 3D object under different growth conditions.
Notice the formation of the lateral bends corresponding to the real nasal capsule shape development from E13.5 to E14.5 occur only in the condition with slow growing purple zones. These bends form …
