Effect of localization, length and orientation of chondrocytic primary cilium on murine growth plate organization

Abstract

The research investigates the role of the immotile chondrocytic primary cilium in the growth plate. This study was motivated by (i) the recent evidence of the mechano-sensorial function of the primary cilium in kidney tubule epithelial cells and (ii) the distinct three-dimensional orientation patterns that the chondrocytic primary cilium forms in articular cartilage in the presence or the absence of loading. For our investigation, we used the Smad1/5(CKO) mutant mouse, whose disorganized growth plate is due to the conditional deletion of Smad 1 and 5 proteins that also affect the so-called Indian Hedgehog pathway, whose physical and functional topography has been shown to be partially controlled by the primary cilium. Fluorescence and confocal microscopy on stained sections visualized ciliated chondrocytes. Morphometric data regarding position, orientation and eccentricity of chondrocytes, and ciliary localization on cell membrane, length and orientation, were collected and reconstructed from images. We established that both localization and orientation of the cilium are definite, and differently so, in the Smad1/5(CKO) and control mice. The orientation of the primary cilium, relative to the major axis of the chondrocyte, clusters at 80° with respect to the anterior-posterior direction for the Smad1/5(CKO) mice, showing loss of the additional clustering present in the control mice at 10°. We therefore hypothesized that the clustering at 10° contains information of columnar organization. To test our hypothesis, we prepared a mathematical model of relative positioning of the proliferative chondrocytic population based on ciliary orientation. Our model belongs to the category of "interactive particle system models for self-organization with birth". The model qualitatively reproduced the experimentally observed chondrocytic arrangements in growth plate of each of the Smad1/5(CKO) and control mice. Our mathematically predicted cell division process will need to be observed experimentally to advance the identification of ciliary function in the growth plate.

Figure 1

The primary cilium of proliferative chondrocytes of wild type mouse at the end of gestation by confocal microscopy. (a) The chondrocytic cilia are visible by monoclonal anti-acetylated-α-tubulin (in red). For the yellow portion of (a), we show in (b) the chondrocytic nucleus stained with DAPI (blue), and in (b) and (c) the endogenous fluorescence is used to visualize the background (darker green).

Figure 2

Proximal femur’s growth plate of double knockout mouse Smad1/5^CKO and WT littermate. (a) Alcian blue-stained sections show lack of chondrocytic columnar arrangements in Smad1/5^CKO, present in WT mouse. Ectopic cartilage is present in the enlarged perichondrium of the mutant (see also Fig. 4 of Retting et al., 2009). (b) Fluorescent microscopy images show primary cilia (red) and nuclei (blue) of chondrocytes that appear lighter than the surrounding ECM.

Figure 3

Localization, length and orientation of primary cilium and orientation of chondrocytes with respect to the medial-lateral axis. We offer examples (diagrams not to scale) of ciliated cells to clarify distinct concepts and appearances in 2D and 3D. The cilium appears shorter in (a) 2D than in (b) 3D. Such length may or may not be the same as the cilia in (c), (d), and (e) that we visualize in 2D only. In 2D, cilia (c) and (d) show same orientation and different localization on the membrane; while (c) and (e) show same localization and different orientation. (f) The Smad1/5^CKO mutants show a wide range of values (green), indicating that the major axis of the chondrocyte can form any angle (between 0° and 180°) with the medio-lateral direction. In comparison, the major axis of proliferative chondrocytes of the WT mice (blue) cluster at values 0° and 180°, indicating that for normal animals, the major axis of the chondrocyte in the proliferative zone is somewhat parallel to the medial-lateral direction. (g) The white lines through the cells denote cellular orientation in the medial-lateral-longitudinal plane.

Figure 4

Definite patterns of ciliary orientation in 3D. The distribution of values for the azimuth angle of the primary cilium (see circumferential ϕ_ci,ce) with respect to its chondrocyte differs between the Smad1/5^CKO and WT mice. One of the values of clustering (ϕ_ci,ce =10°) for WT is lost for the Smad1/5^CKO. We hypothesize that the clustering at 10° controls columnar arrangement in the WT, lost in the Smad1/5^CKO. The other angle θ_ci,ce that together with ϕ_ci,ce defines the orientation of the primary cilium assumes random values (dots span radially the whole 0° to 360° range) for both Smad1/5^CKO and WT.

Figure 5

Mathematical modeling on the basis of experimental data. This diagram illustrates the input and output variables of the equations that define the simulation of cell organization after cell division; and the experimental data used for ciliated chondrocyte representation.

Figure 6

Dynamic simulations of cell positioning after division. We show three steps of the dynamic simulation, when cell division produces a total of 80,120, 160 cell models for Smad1/5^CKO and 60, 90 and 120 cell models for WT. The experimental data were used to represent cell eccentricity, ciliary length and azimuth angle θ_ci,ce.

Figure 7

Distribution patterns of cell centers. Both the distributions obtained from the experimental data and from the results of the cell division simulation show a difference in center distribution between Smad1/5^CKO and WT: isotropy vs. longitudinal anisotropy.

Figure 8

Measure of anisotropy of chondrocytic distributions. (a) We measured the angle θ_nn between nearest neighbor cells relative to the longitudinal direction. (b) A delta distribution at θ_nn = 0° corresponds to complete longitudinal alignment, whereas at θ_nn = 90° corresponds to medial-lateral alignment. A uniform distribution corresponds to complete disorder in cellular alignment. (c) The Smad1/5^CKO cells displayed approximately uniform alignment of nearest neighbors, showing lack of preferential longitudinal orientation. Instead, (d) the WT cells were strongly aligned in the longitudinal direction.

Figure 9

Our dynamic simulation applied to the Col2a-Cre;Kif3a^fl/fl mouse. By assuming only the few cilia present in the Col2a-Cre;Kif3a^fl/fl mouse, our dynamic model computes a weak cell-to-cell signal that impedes the “rotation” lacking in such mouse growth plate where chondrocytes do not align in columns (compare to figure 7c in 5).