In vivo screen for osteoclast derived osteogenic factors using non-resorptive osteoclasts in zebrafish bone

Bone homeostasis requires the balanced action of bone forming osteoblasts and bone resorbing osteoclasts. This balance is maintained through local as well as systemic factors. Loss of osteoclast resorptive activity leads to development of osteopetrosis with increased bone volume but reduced skeletal integrity. Identification of the factors that regulate such anabolic acitivty is hampered by the essential function of these genes in development as well as the inaccessibility of large numbers of primary osteoclasts for biochemical analyses. Here we generated zebrafish with non-resorptive osteoclasts through CRISPR-Cas9 mediated knockout of chloride voltage-gated channel 7 (clcn7). Clcn7 mutants are adult viable, show increased vertebral bone thickness and increased bone formation. Using this in vivo model of persistent, but deficient osteoclasts, we used differential gene expression to identify putative anabolic bone regulators. Transcriptional profiling of clcn7 mutant primary osteoclasts identified differential expression of genes involved in chemotaxis and cytokine signaling. Global transgenic overexpression of a subset of the upregulated, secreted genes showed positive effects of the cytokines csf3b, cxcl19 and the glycosylated peptide progranulin 1 on skeletal parameters, while the cytokine ccl35.1 negatively impacted bone volume. Our findings suggest that the concerted function of these cytokines plays an important role in the regulation of bone homeostasis and that individual cytokines cause distinct changes in vertebral bone form and differentiation.