Fishing for answers: Investigating Fibrodysplasia Ossificans Progressiva using a novel adult zebrafish model
Abstract: Fibrodysplasia Ossificans Progressiva (FOP) is a rare, autosomal
dominant genetic disorder in humans characterized by the gradual ossification of fibrous
tissues, including skeletal muscle, tendons, and ligaments. In humans, activating mutations
in the Type I BMP/TGFβ family member receptor, ACVR1, are associated with FOP. Zebrafish
acvr1l, previously known as alk8, is the functional ... read moreortholog of human ACVR1. The objective
of this work is to create and characterize the first adult zebrafish model for FOP,
providing a useful tool to study the development and progression of FOP-like symptoms.
Constitutively active mutations in zebrafish acvr1l cause early lethal defects. Therefore,
to study roles for activating acvr1l mutations in adult zebrafish, gateway cloning was used
to create a vector containing the hsp70l heat shock promoter driving the expression of
mCherry-tagged constitutively active Acvr1l (Q204D). Constructs were injected into single
cell stage BMP response element reporter (Bre:GFP) zebrafish to create stable Tg(Bre:GFP);
Tg(hsp70l:acvr1l_Q204D-mCherry) lines. Beginning at 2 weeks post fertilization,
developmentally staged transgenic animals were subjected to daily one-hour heat shock
treatments (3 weeks to 12 months) to induce Acvr1lQ204D expression. Micro-CT was used on
whole animals to confirm FOP-like skeletal defects. Acvr1lQ204D-expressing animals
displayed HO formation, abnormal spinal curvature, vertebral fusions, and malformation of
pelvic fins, as compared to heat shocked Tg(Bre:GFP) animals and non-heat shocked controls.
Histological stains (Safranin O and Hall and Brunt's Quadruple Stain) were used to confirm
the presence of cartilaginous proteoglycans and mineralized tissue formation in the HO
lesions. As injury is a known trigger for HO development in human FOP patients, several
injury models were tested on Acvr1lQ204D-expressing animals to induce the formation of
reliable, replicable HO. Activin A injection, cardiotoxin injection, and caudal fin clip
injuries were all developed. None of these methods resulted in HO formation at the site of
injury. However, cardiotoxin injected and caudal fin clipped animals did develop HO at
distant sties, including the body cavity and along the spine. In summary, these results
suggest that heat-shocked Acvr1lQ204D-expressing adult zebrafish provide an informative
model for human FOP, but further work is needed to identify an effective method for
inducing reliable site-directed HO.
Thesis (Ph.D.)--Tufts University, 2018.
Submitted to the Dept. of Cell, Molecular & Developmental Biology.
Advisor: Pamela Yelick.
Committee: Leif Oxburgh, Clifford Rosen, Laura Liscum, and Aris Economides.
Keywords: Biology, Molecular biology, and Genetics.read less