Abstract
Background
The cartilaginous growth plate (GP) is a zonal structure, in which chondrocytes are organized into columns and drive the longitudinal elongation of the endochondral bones. In the proliferative zone (PZ), cells exhibit high mitotic activity, are flattened and oriented along the mediolateral (ML) axis of the GP. Mitotic figures in the elongated chondrocytes lie perpendicular to the proximo-distal (PD) direction of growth, while cell divisions occur parallel to the columns followed by a gliding movement of the daughter cells. The mechanisms responsible for the geometrical anisotropy and columnar arrangement of PZ chondrocytes are poorly understood. Here, we assessed the function of the adhesive receptor β1 integrins on spindle and division geometry in chondrocytes using mouse genetics.
Methods
GP slices were prepared from wild type (wt) and β1fl/fl-Col2a1cre mice. Primary rib chondrocytes were cultured on substrate-coated glass slides and fluorescently stained with anti-alpha-tubulin and anti-pericentrin antibodies, with phalloidin and DAPI. Confocal stacks were built from images acquired by confocal microscopy. Cell and spindle orientation relative to the PD axis (in vivo) or to the substrate plane (SP) in vitro were determined by geometric functions. Shape indexes (SI) were calculated as the ratio of the length and height of the cell.
Results
During GP morphogenesis, aggregating mesenchymal cells (E11) were polygonal (SI=1.43) and nonoriented. Upon chondrogenic differentiation at E12, wt GP chondrocytes showed moderate flattening (SI=1.93) and tend to align perpendicular to the PD axis. At E13, PZ chondrocytes further flattened (SI=3.41) and largely organized into lines along the ML axis. At E14, the first vertical stacks formed, which were gradually elongated along the PD axis at later stages and composed of extremely flat (SI=4.91), ML-oriented chondrocytes. Early spindles were randomly oriented at all stages, whereas late spindles were aligned along the long axis of the cell. In contrast, β1 integrin null PZ chondrocytes were rounded (SI=1.37), displayed random orientation of both early and late spindles, and failed to organize into columns. On collagen II, both wt and β1-null primary chondrocytes remained rounded and displayed random spindle orientation relative to the SP. On fibronectin (FN), wt chondrocytes were flattened (SI=3.26) and their spindle was aligned parallel to SP. Mutant cells lacking the major FN receptor α5β1 integrin did not spread, were rounded (SI=1.73) and aligned their spindle randomly. On vitronectin, both cell types spread and flattened well (SI=4.11 and SI=4.20) accompanied by parallel spindle orientation to the SP.
Conclusion
We propose that β1 integrins provide adhesive cues for chondrocyte geometry and orientation, which in turn orients the mitotic spindle and determines the division axis; and for chondrocyte intercalation to shape the three-dimensional structure of the GP.
