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

ABSTRACT: Bone homeostasis requires the balanced action of bone forming osteoblasts and bone resorbing osteoclasts and 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. Work on osteopetrotic mouse models has identified a role for osteoclast released factors in the regulation of bone anabolism, but further analysis of these factors is hampered by the general essential function of osteopetrosis genes in development and the inability to isolate primary osteoclasts in sufficient numbers. Here we generated a zebrafish osteopetrosis model through CRISPR mediated knockout of chloride voltage-gated channel 7 (clcn7) which is adult viable and shows increased vertebral bone thickness. Isolation of primary osteoclasts from wildtype and clcn7 mutants by FACS followed by transcriptional profiling, identified upregulation of genes involved in chemotaxis and cytokine signaling in clcn7 deficient osteoclasts. Global transgenic overexpression of a subset of the upregulated genes further identified distinct, but positive effects of the cytokines csf3b, cxcl19 and the glycosylated peptide progranulin 1 (grn1) 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 affect regional areas and distinct parameters of vertebral bone form and differentiation.

ORGANISM(S): Danio rerio

PROVIDER: GSE308663 | GEO | 2026/08/05

REPOSITORIES: GEO

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Project description:Enhanced osteoclastogenesis and osteoclast activity contribute to the development of osteoporosis, which is characterized by increased bone resorption and inadequate bone formation. As novel anti-osteoporotic therapeutics are needed, understanding the genetic regulation of human osteoclastogenesis could help identify potential treatment targets. This study aimed to provide an overview of the transcriptional reprogramming during human osteoclast differentiation. Osteoclasts were differentiated from CD14+-monocytes from eight female donors. RNA-sequencing during differentiation demonstrated 8980 differentially expressed genes grouped into eight temporal patterns conserved across donors. These patterns showed distinct molecular functions, associated with postmenopausal osteoporosis susceptibility genes based on RNA from iliac crest biopsies, and bone mineral density SNPs. Network analyses showed mutual dependencies between the temporal expression patterns and provides insight into subtype-specific transcriptional networks. Donor specific expression patterns identified genes at monocyte stage, such as filamin B (FLNB) and oxidized low density lipoprotein receptor 1 (OLR1, encoding LOX-1), that are predictive for the resorptive activity of mature osteoclasts. Differentially expressed G-protein coupled receptors showed strong expression during osteoclast differentiation and associated with bone mineral density SNPs, implying a pivotal role in osteoclast differentiation and activity. The regulatory effects of three differentially expressed G-protein coupled receptors were exemplified by in vitro pharmacological modulation of complement 5A receptor 1 (C5AR1), somatostatin receptor 2 (SSTR2), and free fatty acid receptor 4 (FFAR4/GPR120). Activating C5AR1 enhanced osteoclast formation, while activating SSTR2 decreased resorptive activity of mature osteoclasts, and activating FFAR4 decreased both number and resorptive activity of mature osteoclasts. In conclusion, we report the transcriptional reprogramming during human osteoclast differentiation and identified SSTR2 and FFAR4 as anti-resorptive G-protein coupled receptors as well as FLNB and LOX-1 as potential molecular markers of osteoclast activity. These data can help future investigations to identify molecular regulators of osteoclast differentiation and activity and provide the basis for novel anti-osteoporotic targets.

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

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