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 Smad1/5CKO 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 Smad1/5CKO 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 Smad1/5CKO 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 Smad1/5CKO 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.

Automated Cell Detection and Morphometry on Growth Plate Images of Mouse Bone

“…Further, the mean isoperimetric ratio IR is significantly different between resting and proliferative zones, and between proliferative and hypertrophic zones, indicating that the cells become less circular as they progress from the resting to proliferative, and from proliferative to hypertrophic zone. Furthermore, our CDA confirms the rounder shape, and the orientation off the horizontal (medial-lateral) direction, of chondrocytes in the Smad1/5 CKO mutant in comparison with the control WT mouse (Figure 3), previously determined by manual detection [10]. Note that for the CDA analysis of this WT growth plate, the Retinex parameter NI was set to zero because these fluorescent microscopy images showed no faint cells behind the plane of focus.…”

“…[10], [11]). Longitudinal growth of bones is the result of a process involving cell division, migration, and then ossification, that occurs in growth plates located at both the proximal and distal ends of the long bone.…”

Primary Cilia and Coordination of Receptor Tyrosine Kinase (RTK) and Transforming Growth Factor β (TGF-β) Signaling

“…As an example, inhibition of Ciliary RTK and TGF-b Signaling Cite this article as Cold Spring Harb Perspect Biol 2017;9:a028167 SMAD2 signaling reduces the length of motile cilia at the gastrocoel roof plate, at the Xenopus left -right (LR) organizer, and at the neural tube and the epidermis, and blockage of TGF-b signaling was suggested to impair the structure and/or function of the ciliary TZ as evidenced by lack of the TZ protein B9D1/MSKR-1 in cilia emerging from the epidermis (Tozser et al 2015). In chondrocytic cells, TGF-b signaling was reported to suppress the levels of Ift88 mRNA stability, leading to a reduced average length and number of primary cilia (Kawasaki et al 2015), and in the disorganized growth plate of Smad1/5 CKO mutant mice, the orientation of projecting primary cilia is disturbed (Ascenzi et al 2011). These results suggest that TGF-b signaling plays multiple roles in cilia, and future studies should focus on the mechanisms by which the CiPo is organized and regulated to control endo-and exocytic events in ciliary signaling, and how TGF-b signaling itself impacts on ciliary length and TZ organization.…”

Axonemal positioning and orientation in three‐dimensional space for primary cilia: What is known, what is assumed, and what needs clarification

“…Given that inappropriate positioning and/or 3-D orientation of cilia is a characteristic of ciliopathies of sensory and motile cilia, including nodal cilia, it is not surprising that a similar loss of orientation/position is found in ciliopathies of the primary cilium (referred to as Primary Primary Ciliary Dyskinesia ( sic ) by Wheatley,2005) that have a skeletal phenotype (McGlashan et al,2007; Ruiz-Perez et al,2007; Haycraft and Serra,2008; Nigg and Raff,2009; Ochiai et al,2009; Retting et al,2009; Zaghloul and Katsanis,2009; Lee et al,2010; Qiu et al,2010; Ascenzi et al,2011; Zaghloul and Brugman,2011). The best studied of these at the cellular level have been in growth plate cartilage, which functions during both prenatal and postnatal life as the tissue responsible for elongation of bones.…”

“…That is, there is an interrelationship between loss of appropriate axonemal positioning characteristics and compromised ciliary function. One approach to address this question is through modeling axonemal positional characteristics in normal mice compared to mutants with skeletal phenotypes (Ascenzi et al,2011). The larger challenge is to develop experimental systems to analyze the molecular mechanisms linking compromised function of primary cilia in connective tissues to inappropriate axonemal positioning characteristics, and to establish the relationship of compromised ciliary function to loss of both tissue anisotropy and appropriate cellular shape.…”