Introduction: The zebrafish Type I BMP/TGFβ family member receptor, Acvr1l, modulates BMP/TGFβ signaling to promote cartilage and bone formation. Activating mutations in the human ortholog of Acvr1l, ACVR1, are associated with Fibrodysplasia Ossificans Progressiva (FOP), a disease characterized by the gradual ossification of fibrous tissues, including skeletal muscle, tendons, and ligaments. The objective of this work is to validate and characterize a zebrafish model for FOP to elucidate the molecular mechanisms driving heterotopic bone formation in FOP and other human diseases.
Methods: Gateway cloning, transgenesis, automated heat shock system, fluorescence imaging, Alcian blue stain, Alizarin Red Stain, μCT, histology, IHC
Results: Gateway cloning was used to create a vector containing the Hsp70 heat shock (HS) promoter driving the expression of mCherry-tagged constitutively active (CA) Acvr1l. Constructs were injected into single cell stage BMP response element reporter (BRE-GFP) zebrafish to create stable transgenic hs-CA-Acvr1l-mCherry::BRE-GFP lines. Developmentally staged transgenic animals were subjected to daily one-hour heat shock treatments (3 weeks to 8 months) to induce CA-Acvr1l expression. An Activin A injection injury model was developed for heat shocked CA-Acvr1l expressing animals to define the time course of HO progression in these animals. We are currently performing μCT and Alcian blue/Alizarin red staining on these animals to characterize mineralization defects and to identify sites of heterotopic ossification (HO) as observed in human FOP patients. In addition, we are performing paraffin embedding and sectioning on selected specimens to perform histological and immunohistochemical analyses. Preliminary results show that CA-Acvr1l-expressing zebrafish exhibit enhanced/accelerated mineralization, vertebral fusions, osteochondroma formation, and HO phenotypes, as compared to HS WT and non-HS CA-Acvr1l transgenic zebrafish controls. IHC analyses of activated BMP/TGFβ signaling (pSmad1/5, pSmad2/3), and chondrocyte (Collagen II, Sox9), endothelial cell (Tie2, vWF) and osteoblast differentiation markers (Runx2, Osteocalcin) are being used to characterize these defects on a cellular level.
Conclusions: HS-inducible CA-Acvr1l transgenic zebrafish provide an informative model for HO development in human FOP.