Fishing for answers: Investigating Fibrodysplasia Ossificans Progressiva using a novel adult zebrafish model
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