Intramembranous ossification (bone formation within connective tissue), endochondral ossification (bone formation via mineralization and reabsorption of cartilage anlagen) and apoptotic remodeling of unmineralized cartilage (chondroptosis) are mechanisms underlying skeletal tissue morphogenesis. Chondroptosis, a chondrocyte specific form of apoptosis, is important for cartilage morphogenesis and is altered in some diseases, such as osteoarthritis. Nevertheless, the molecular basis remains elusive because chondrocytes undergo classical apoptosis in culture and in vivo models are lacking.
Here we describe a conserved role for the motor protein Kinesin I, kif5B, in maintaining muscle fiber integrity, and a novel role in apoptosis-mediated remodeling of cartilage. In addition to the hallmark features of chondroptosis (expansion of rER and Golgi, increased autophagocytic vacuoles, and patchy chromatin), extrusion and engulfment of mutant chondrocytes by neighboring cells occurs in kif5B loss of function (kif5Blof) mutants and chimeric contexts. Genetic interactions with Wnt planar cell polarity (PCP) mutants indicate distinct roles for PCP and kif5B in cartilage morphogenesis. Initial establishment of the cartilage is unaffected as early chondrocytic markers (sox9 and col2α1) are properly expressed. Later, between 60 and 72hpf, when cartilage remodeling occurs, kif5Blof mutants undergo prolonged chondroptosis. Chimeric analyses, using cell transplantation, indicate a cell-autonomous requirement for Kif5B in regulating secretion, nuclear position, cell elongation and survival. Surprisingly, in these assays large groups of wild-type cells could support elongation of neighboring mutant cells. Mosaic expression of kif5Ba but not kif5Aa, in cartilage using Gal4/UAS similarly rescues the chondroptosis phenotype.
Metalloproteinases are crucial for cartilage remodeling before mineralization, and the cartilage phenotypes of MT1-MMP mutant mice resemble those of kif5Blof zebrafish. Moreover, Kif5B mediates membrane localization of MT1-MMP in other contexts; therfore, Kif5B may promote MT1-MMP functions in cartilage remodeling. To circumvent the earlier phenotypes reported for MT1-MMPa knockdown in zebrafish we utilized a pharmacological approach. However, general inhibition of metalloproteinase activity (EDTA) or more specific inhibition of MMP activity (GM6001) has failed to phenocopy kif5Blof mutants, and thus MT1-MMP contribution remains ambiguous. Cumulatively, our study reveals an essential role for Kif5B in cell polarization, secretion, and chondroptosis. kif5Blof mutants provide a new in vivo model to study a poorly understood form of programmed cell death that contributes to cartilage remodeling and osteochondrodysplasia in humans.