Kinesin-1 promotes chondrocyte maintenance during skeletal morphogenesis

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.

ClC-7 Regulates the Pattern and Early Development of Craniofacial Bone and Tooth

“…The efficiency of MO was evaluated by measuring the expression of clcn7 as assessed by Q-PCR and phenotypic penetrance. The injected embryos were cultured in the embryo medium and harvested at 3 dpf (days post-fertilization) or 5 dpf for the following experiments 28.…”

Zebrafish as a model to study autophagy and its role in skeletal development and disease

“…Together, these tools provide a more accurate way to measure autophagy activity and differences in autophagy levels quantitatively between different fish. Indeed, these tools have been used in zebrafish studies to determine how autophagic flux is affected by specific genetic mutations within bone and cartilage cells (Hu et al 2019 ; Santos-Ledo et al 2017 ). These studies showed that changes to bone and cartilage cell differentiation and functioning were in part due to dysregulated autophagy activity.…”

Ciliary kinesins beyond IFT: Cilium length, disassembly, cargo transport and signalling

“…Invalidation of KIF5B in the mouse is embryonic lethal, although whether the mutant embryos presented with ciliopathy‐related phenotypes was not described [Tanaka et al ., ]. In Zebrafish, KIF5B isoforms were involved in cartilage remodelling and maintenance during craniofacial morphogenesis, but without an obvious connection to cilia‐related function [Santos‐Ledo et al ., ]. Loss of KLC1 in the mouse leads to brain specific defects which were not related to cilia [Rahman et al ., ].…”