Zebrafish models for human FKRP muscular dystrophies

Abstract

Various muscular dystrophies are associated with the defective glycosylation of α-dystroglycan and are known to result from mutations in genes encoding glycosyltransferases. Fukutin-related protein (FKRP) was identified as a homolog of fukutin, the defective protein in Fukuyama-type congenital muscular dystrophy (FCMD), that is thought to function as a glycosyltransferase. Mutations in FKRP have been linked to a variety of phenotypes including Walker–Warburg syndrome (WWS), limb girdle muscular dystrophy (LGMD) 2I and congenital muscular dystrophy 1C (MDC1C). Zebrafish are a useful animal model to reveal the mechanism of these diseases caused by mutations in FKRP gene. Downregulating FKRP expression in zebrafish by two different morpholinos resulted in embryos which had developmental defects similar to those observed in human muscular dystrophies associated with mutations in FKRP. The FKRP morphants showed phenotypes involving alterations in somitic structure and muscle fiber organization, as well as defects in developing eye morphology. Additionally, they were found to have a reduction in α-dystroglycan glycosylation and a shortened myofiber length. Moreover, co-injection of fish or human FKRP mRNA along with the morpholino restored normal development, α-dystroglycan glycosylation and laminin binding activity of α-dystroglycan in the morphants. Co-injection of the human FKRP mRNA containing causative mutations found in human patients of WWS, MDC1C and LGMD2I could not restore their phenotypes significantly. Interestingly, these morphant fish having human FKRP mutations showed a wide phenotypic range similar to that seen in humans.

Drug screening in a zebrafish model of Duchenne muscular dystrophy

“…Abnormal birefringence of muscle was observed and analyzed by placing anesthetized embryos on a glass-polarizing filter and subsequently covering them with a second polarizing filter as described in ref. 18. Screening the Prestwick Library for Compounds That Decrease the Percentage of Affected Zebrafish.…”

“…The morpholino sequences for dystrophin were MO: 5′-TTGAGTCCTTTAATCC-TACAATTTT-3′; 6 ng morpholinos were injected into the yolk of one-to twocell stage embryos as described in ref. 18. At 1 dpf, 20 injected embryos were arrayed in a 24-well plate and were treated with individual chemicals (1-7).…”

“…Immunohistochemical staining of wholemutant and morphant fish bodies was done as described in ref. 18. Embryos were incubated separately with anti-dystrophin (1:25; Sigma) or anti-laminin (1:25; Sigma) antibodies at 4°C overnight.…”

“…After washing three times, sections were incubated with secondary antibodies and examined as described in ref. 18.…”

“…In addition to their suitability for chemical screens, zebrafish also represent a good model to investigate genes involved in muscle development and degeneration, including human muscular dystrophy (12)(13)(14)(15)(16)(17)(18). The orthologs of many dystrophin-glycoprotein complex (DGC) components are expressed in zebrafish (19).…”