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Understanding the Mechanisms Driving Ectopic Bone Formation in a Zebrafish FOP Model
Author(s) -
LaBonty Melissa,
Yelick Pamela
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.216.5
Subject(s) - zebrafish , fibrodysplasia ossificans progressiva , microbiology and biotechnology , ectopic expression , chemistry , biology , cell culture , anatomy , genetics , ossification , biochemistry , gene
The zebrafish Type I TGFβ family member receptor, Alk8, modulates BMP/TGFβ signaling to promote cartilage and bone formation. Activating mutations in the human ortholog of Alk8, ACVR1 (ALK‐2), are associated with the disease Fibrodysplasia Ossificans Progressiva (FOP), characterized by the gradual ossification of fibrous tissues. The objective of this study is to develop a zebrafish model to identify and characterize molecular mechanisms underlying FOP. Methods: Gateway cloning, transgenesis, Alcian blue stain, Alizarin Red Stain, μCT, IHC Results: Gateway cloning was used to generate Tol2 vectors containing a heat shock promoter driving the expression of mCherry tagged wild type (WT), dominant negative (DN), or constitutively active (CA) Alk8. The Tol2 vectors were co‐injected with Tol2 transposase mRNA into single cell staged BMP response element reporter (BRE:GFP) zebrafish to produce transgenic animals. Transgenic lines were subjected to short term (1‐3 weeks) or long term (1‐3 months) daily heat shock intervals to induce variant Alk8 expression. μCT, Alcian blue, and Alizarin red staining were used to characterize changes in mineralization patterns. CA‐Alk8‐expressing zebrafish exhibited enhanced mineralization and ectopic bone formation as compared to WT and non‐heat shock controls. In contrast, DN‐Alk8‐expressing animals exhibited reduced mineralization. IHC analyses of phosphorylated SMADs and other BMP and TGFβ downstream targets are being used to identify the molecular and cellular mechanisms driving aberrant mineralization. Conclusions Heat shock inducible Alk8 variants in Zebrafish provide informative models for ectopic bone formation in human FOP. Support: NSF GRFP NS9344 (ML), NIH/NIDCR R01 DE016962 (PCY), R01 DE018043 (PCY), and R21 HD085013 (PCY).