Cell Division And Rearrangement Dynamics Coordinated By Planar Cell Polarity Pathway In Growth Plate Cartilage

Chondrodysplasias, resulting in dwarfism and altered skeletal growth, are heritable disorders affecting 1 in 4000 to 5000 births. Long bone growth is tightly controlled by sequential chondrocyte maturation through spatially distinct zones in growth plate cartilage. Discoid proliferative chondrocytes form a division plane parallel to the longitudinal axis of the bone and rotate 90 degrees to stack the cells in a unique columnar architecture, which potentiates further limb growth. Noncanonical, β-catenin-independent, Wnt signaling is needed to align division planes and promote organized column formation, which makes the planar cell polarity (PCP) pathway a strong candidate in determining column architecture. Viral manipulation of PCP pathway proteins in chicken embryo growth plates interfered with cell division orientation, but conclusions were limited by fixed tissue sections. To expand our depth of mechanistic inquiry, we sought to develop a method to analyze genetic deletions of PCP pathway proteins and dynamically monitor chondrocyte rearrangement in living growth plate tissue. We hypothesize that Wnt-dependent PCP signaling is required for proliferative chondrocyte rearrangement. Using mice with membrane bound GFP expression and confocal microscopy, we generated time-lapse videos of chondrocytes from live cranial base growth plate explants. Genetic knockout of upstream Wnt5a ligand and inducible, cartilage-specific deletion of Vangl2 transmembrane receptor and Ror2 kinase co-receptor result in significant differences in the final division plane angle distributions compared to normal (p-value<0.0001). Vangl2 is a core PCP protein phosphorylated by Ror2 in response to Wnt5a signaling. While disrupted rearrangement could be a cause of the disorganized proliferative zone, the daughter cells also have premature separation at their interface. Normal chondrocytes separate on average 14.0±6.6 hours after division, while Ror2, Wnt5a, and Vangl2 mutants have significantly shorter separation times (5.2±4.0, 7.8±4.4, and 9.1±3.9 hours, respectively). Our results show that proper rearrangement of proliferative chondrocytes is dependent on PCP pathway activity; however, it is unclear if the premature separation is occurring through disengagement of the cadherin-dependent daughter cell interface or deposition of extracellular matrix between the cells. The focus of future studies is to determine what is acting downstream of the PCP pathway to regulate adhesion dynamics and determine the extrinsic requirements guiding polarized chondrocyte behaviors. These results will advance approaches to cartilage engineering to treat chondrodysplasias by understanding the mechanistic cues required for proper cartilage tissue architecture and growth.