Abstract
During skeletal morphogenesis diverse mechanisms are used to support bone formation. This can be seen in the bones that require a cartilage template for their development. In mammals the cartilage template is removed, but in zebrafish the cartilage template persists and the bone mineralizes around the cartilage scaffold. Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements; however, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear. We report double mutants disrupting two zebrafish kinesin-I genes (hereafter kif5Blof) that we generated using CRISPR/Cas9 mutagenesis. We show that zygotic Kif5Bs have a conserved function in maintaining muscle integrity, and are required for cartilage remodeling and maintenance during craniofacial morphogenesis by a PCP-distinct mechanism. Further, kif5Blof does not activate ER stress response genes, but instead disrupts lysosomal function, matrix secretion, and causes deregulated autophagic markers and eventual chondrocyte apoptosis. Ultrastructural and transplantation analysis reveal neighboring cells engulfing extruded kif5Blof chondrocytes. Initial cartilage specification is intact; however, during remodeling, kif5Blof chondrocytes die and the cartilage matrix devoid of hypertrophic chondrocytes remains and impedes normal ossification. Chimeric and mosaic analyses indicate that Kif5B functions cell-autonomously in secretion, nuclear position, cell elongation and maintenance of hypertrophic chondrocytes. Interestingly, large groups of wild-type cells can support elongation of neighboring mutant cells. Finally, mosaic expression of kif5Ba, but not kif5Aa in cartilage rescues the chondrocyte phenotype, further supporting a specific requirement for Kif5B. Cumulatively, we show essential Kif5B functions in promoting cartilage remodeling and chondrocyte maintenance during zebrafish craniofacial morphogenesis.
Highlights
Remodeling of unmineralized cartilage occurs via planar cell polarity (PCP) mediated cell rearrangements that contribute to lengthening of elements
We identified a conserved role for the Kinesin-1 heavy chain, kif5B, in maintaining muscle integrity and a novel role in cartilage development during craniofacial morphogenesis
When kif5B is lost, autophagic markers are deregulated leading to eventual chondrocyte apoptosis
Summary
Intramembranous ossification–formation of bone within connective tissue, and endochondral or perichondral ossification–formation of bone via mineralization and reabsorption of cartilage anlage (endochondral) or mineralization around a persisting cartilage scaffold (perichondral) that serve as templates for later bone formation, and apoptotic remodeling of unmineralized cartilage are mechanisms that contribute to skeletal tissue morphogenesis [1]. During morphogenesis the chondrocytes of long bones, endochondral or perichondral bones in teleost fish, including those in the jaw, elongate and orient their primary cilia and microtubule organizing centers (MTOC), and, via oriented intercalation, the discoid cells align in a stacked configuration [3]. Unmineralized cartilage remodels via conserved PCP-mediated polarization and cell rearrangements, the mechanisms that maintain the chondrocyte template that supports perichondral ossification remain unclear
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