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Zebrafish models for human FKRP muscular dystrophies.

Kawahara G, Guyon JR, Nakamura Y, Kunkel LM - Hum. Mol. Genet. (2009)

Bottom Line: 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.Additionally, they were found to have a reduction in alpha-dystroglycan glycosylation and a shortened myofiber length.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Program in Genetics, Harvard Medical School, Children's Hospital, Boston, MA, USA.

ABSTRACT
Various muscular dystrophies are associated with the defective glycosylation of alpha-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 alpha-dystroglycan glycosylation and a shortened myofiber length. Moreover, co-injection of fish or human FKRP mRNA along with the morpholino restored normal development, alpha-dystroglycan glycosylation and laminin binding activity of alpha-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.

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Head and body in FKRP morphant at 4 dpf. (A and B) wild-type fish. (C and D) Control MO1 injected fish. (E and F) Control MO2 injected fish. (G and H) FKRP MO1 injected fish. (I and J) FKRP MO2 injected fish. (A), (C), (E), (G) and (I) show bright images. (B), (D), (F), (H) and (J) show results of birefringence assay. Arrows show smaller eyes in affected fish. Arrowheads show inflated heart in affected fish. Bar: 100 µm.
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DDP528F1: Head and body in FKRP morphant at 4 dpf. (A and B) wild-type fish. (C and D) Control MO1 injected fish. (E and F) Control MO2 injected fish. (G and H) FKRP MO1 injected fish. (I and J) FKRP MO2 injected fish. (A), (C), (E), (G) and (I) show bright images. (B), (D), (F), (H) and (J) show results of birefringence assay. Arrows show smaller eyes in affected fish. Arrowheads show inflated heart in affected fish. Bar: 100 µm.

Mentions: FKRP morphant embryos lacked locomotor activity and did not respond to touch with the normal escape response observed in control embryos. Overall, FKRP morphants appeared to have a smaller eye size compared with controls (arrows in Fig. 1G and I) and many had inflated precardia (arrowheads in Fig. 1G and I). FKRP morphant embryos were also found to have an irregular arrangement of muscle fibers compared with control embryos (arrows in Fig. 2G and I). To better visualize the structure and organization of muscle fibers of the morphants, FKRP morphant and control embryos at 4 dpf were analyzed by birefringence assays (Figs 1 and 2). FKRP morphant embryos were found to have markedly reduced normal patterns of birefringence compared with control (Figs 1H and 1J and 2H and I). To confirm the knock down of the expression of fish FKRP with morpholino injection, endogenous FKRP protein was analyzed with antibodies against fish FKRP. In control MO1 and MO2, the FKRP directed antibody recognized 60 kDa protein on western blots, a size predicted by the fish FKRP sequence. The analysis of morphant 1 and 2 extracts showed that the expression of FKRP protein in morphants was substantially decreased compared with those of control MO1- and MO2-injected fish (Fig. 2K). Western blot results show that the morpholinos used in these injections were effective in knocking down the expression of FKRP during zebrafish development.


Zebrafish models for human FKRP muscular dystrophies.

Kawahara G, Guyon JR, Nakamura Y, Kunkel LM - Hum. Mol. Genet. (2009)

Head and body in FKRP morphant at 4 dpf. (A and B) wild-type fish. (C and D) Control MO1 injected fish. (E and F) Control MO2 injected fish. (G and H) FKRP MO1 injected fish. (I and J) FKRP MO2 injected fish. (A), (C), (E), (G) and (I) show bright images. (B), (D), (F), (H) and (J) show results of birefringence assay. Arrows show smaller eyes in affected fish. Arrowheads show inflated heart in affected fish. Bar: 100 µm.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2807370&req=5

DDP528F1: Head and body in FKRP morphant at 4 dpf. (A and B) wild-type fish. (C and D) Control MO1 injected fish. (E and F) Control MO2 injected fish. (G and H) FKRP MO1 injected fish. (I and J) FKRP MO2 injected fish. (A), (C), (E), (G) and (I) show bright images. (B), (D), (F), (H) and (J) show results of birefringence assay. Arrows show smaller eyes in affected fish. Arrowheads show inflated heart in affected fish. Bar: 100 µm.
Mentions: FKRP morphant embryos lacked locomotor activity and did not respond to touch with the normal escape response observed in control embryos. Overall, FKRP morphants appeared to have a smaller eye size compared with controls (arrows in Fig. 1G and I) and many had inflated precardia (arrowheads in Fig. 1G and I). FKRP morphant embryos were also found to have an irregular arrangement of muscle fibers compared with control embryos (arrows in Fig. 2G and I). To better visualize the structure and organization of muscle fibers of the morphants, FKRP morphant and control embryos at 4 dpf were analyzed by birefringence assays (Figs 1 and 2). FKRP morphant embryos were found to have markedly reduced normal patterns of birefringence compared with control (Figs 1H and 1J and 2H and I). To confirm the knock down of the expression of fish FKRP with morpholino injection, endogenous FKRP protein was analyzed with antibodies against fish FKRP. In control MO1 and MO2, the FKRP directed antibody recognized 60 kDa protein on western blots, a size predicted by the fish FKRP sequence. The analysis of morphant 1 and 2 extracts showed that the expression of FKRP protein in morphants was substantially decreased compared with those of control MO1- and MO2-injected fish (Fig. 2K). Western blot results show that the morpholinos used in these injections were effective in knocking down the expression of FKRP during zebrafish development.

Bottom Line: 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.Additionally, they were found to have a reduction in alpha-dystroglycan glycosylation and a shortened myofiber length.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Genetics, Program in Genetics, Harvard Medical School, Children's Hospital, Boston, MA, USA.

ABSTRACT
Various muscular dystrophies are associated with the defective glycosylation of alpha-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 alpha-dystroglycan glycosylation and a shortened myofiber length. Moreover, co-injection of fish or human FKRP mRNA along with the morpholino restored normal development, alpha-dystroglycan glycosylation and laminin binding activity of alpha-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.

Show MeSH
Related in: MedlinePlus