Bone Abstracts

Osteoclasts are multinucleated giant cells derived from the monocyte/macrophage lineage in the presence of receptor activator of nuclear factor kappa-B ligand (RANKL) and colony-stimulating factor 1 (CSF1). The bone remodeling process in zebrafish is incompletely understood. Here we describe several methods to quantify bone formation and resorption using a zebrafish mutant that lacks functional colony stimulating factor 1a receptor (csf1ra^mh5/mh5). Mice deficient in CSF1 receptor (CSF1R) are toothless and severely osteopetrotic due to the absence of osteoclasts. Zebrafish have two csf1r genes, csf1ra and csf1rb. It was shown before, that fish lacking csf1ra exhibit deformed neural and hemal arches, and have a decreased number of osteoclasts during fin regeneration.

To quantify osteoclast number and bone resorption, respectively, we stained scales from csf1ra^mh5/mh5 mutants and WT siblings for tartrate-resistant acid phosphatase (TRAP) and mineralized tissue (Von Kossa). We observed fewer TRAP positive osteoclasts in csf1ra^mh5/mh5 mutants compared to controls (P<0.0001), which we could also see using a transgenic osteoclast reporter line. Additionally, we found that the area of resorbed bone in the scales of mutant fish was reduced (P<0.0001), confirming the reduction in mature osteoclast numbers in the csf1ra^mh5/mh5 mutant. Studying the rate of bone formation in vivo using calcein and alizarin red staining, revealed that csf1ra^mh5/mh5 mutants make bone at a higher rate than controls (P<0.0001). To show that, as in mammals, zebrafish develop an osteoclast-poor osteopetrosis phenotype in the absence of a functional Csf1r, we will further analyze bone structure and density by microCT.

Here we demonstrate that, as in mammals, zebrafish osteoclastogenesis is dependent on csf1 signaling. We developed and validated valuable techniques for studying zebrafish mutants having unbalanced bone remodeling and supporting use of forward genetics in zebrafish for identification of enhancers and suppressors of bone remodeling phenotypes.