NCAD and ITGB1 Coordinately Regulate Growth Plate Cartilage Architecture

Abstract

Spatial and temporal regulation of chondrocyte maturation in the growth plate drives long bone growth. A notable feature of growth plate cartilage is the progression of progenitor resting chondrocytes into clonal columns of transit‐amplifying proliferative chondrocytes. Following division of proliferative chondrocytes, daughter cell pairs undergo a unique 90‐degree rearrangement to form columns. Our previous studies using a murine fluorescent membrane reporter system and time‐lapse imaging along with chemical inhibition of cell adhesion suggested a primary requirement for cadherin adhesion at the daughter cell interface in column formation. Studies designed to test the force generating mechanism at the cadherin dependent daughter cell interface produced a paradoxical result with activated myosin motor proteins enriched at the cell‐matrix interface, an observation that suggests integrin adhesion molecules might drive cell rearrangement. The purpose of the current work was to determine the relative contributions of cadherin and integrin mediated cell adhesion in chondrocyte column formation. As a first step, we used live cell imaging to compare the daughter cell rearrangement phenotype in growth plate explants from embryos containing a cartilage‐specific deletion of N‐cadherin (NCAD) or integrin β1 (ITGB1). Deletion of either gene produces similar disruption of column formation, even though the mutant phenotypes show differences in cell shape, rearrangement dynamics, and kinetics of daughter cell separation following rearrangement. The apparent requirement for both NCAD and ITGB1 suggested potential reciprocal regulatory interaction between the two adhesion surfaces. To test this hypothesis, we analyzed phenotypes of an allelic series. We found that chondrocytes mutant for ITGB1 show defects in cell rearrangement and column formation (n=37 embryos) regardless of the NCAD genotype as expected from single mutant studies. Surprisingly, chondrocytes mutant for NCAD and heterozygous for ITGB1 are phenotypically wildtype with organized columns and normal rearrangement (n=13 embryos). These results support a model for which ITGB1, and not NCAD, is the driver of chondrocyte rearrangement and suggest that failure to rearrange in NCAD mutants results from increased ITGB1 function. Thus, rearrangement requires a narrow range of cell‐matrix adhesion strength and NCAD potentially acts as a brake by restricting ITGB1 activity in non‐rearranging resting chondrocytes. Current studies aim to confirm these genetic findings using gene expression analysis and an innovative quantitative adhesion assay as well as identify upstream regulators that control strength of antagonism between the cell adhesion interfaces.