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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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The gaps between myofibers can be repaired by co-injection of normal FKRP mRNA with MO2 but not all human mutant alleles. Co-immunostaining of 4 dpf fish co-injected FKRP mRNA with anti MHC (green) and anti laminin (red). (A) Control MO2. (B) Human normal FKRP mRNA co-injected fish. (C) FKRP MO2 injected fish. (D) Human mutated FKRP mRNA (L276I) co-injected fish. (E) Human mutated FKRP mRNA (C318Y) co-injected fish. (F) Human mutated FKRP mRNA (A455D) co-injected fish. Bar: 50 µm. In control and human control mRNA co-injected fish, the myofibers are adjacent at myosepta (arrows in A and B); however, FKRP morphant has the gap between myosepta (arrowhead in C). In L276I mutant fish, the gap is also narrowed (arrow in D). In C318Y and A455D mutant fish, there still remain gaps similar to that found in FKRP morphant fish (arrows in E and F).
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DDP528F7: The gaps between myofibers can be repaired by co-injection of normal FKRP mRNA with MO2 but not all human mutant alleles. Co-immunostaining of 4 dpf fish co-injected FKRP mRNA with anti MHC (green) and anti laminin (red). (A) Control MO2. (B) Human normal FKRP mRNA co-injected fish. (C) FKRP MO2 injected fish. (D) Human mutated FKRP mRNA (L276I) co-injected fish. (E) Human mutated FKRP mRNA (C318Y) co-injected fish. (F) Human mutated FKRP mRNA (A455D) co-injected fish. Bar: 50 µm. In control and human control mRNA co-injected fish, the myofibers are adjacent at myosepta (arrows in A and B); however, FKRP morphant has the gap between myosepta (arrowhead in C). In L276I mutant fish, the gap is also narrowed (arrow in D). In C318Y and A455D mutant fish, there still remain gaps similar to that found in FKRP morphant fish (arrows in E and F).

Mentions: In FKRP morphant embryos, the distance between the myosepta was reduced and the gap between myofibers was greater. To examine the nature of the protein structure at the gap, myofibers in mutant fish were examined by immunostaining with anti-MHC and anti-laminin. In control and human normal FKRP mRNA co-injected fish, the myofibers were adjacent at myosepta (Fig. 7A and B). The gaps between myofibers that were found in FKRP morphants (Fig. 7C) were also reduced with the injection of normal human FKRP mRNA (Fig. 7B). The length of myofibers was of similar size to that seen in control (Fig. 7A). Using the L276I mutant allele, the gap was reduced compared with FKRP morphants alone and the other two mutant alleles, C318Y and A455D (Fig. 7D). In C318Y and A455D mutant alleles, the gap was still wide and myofibers were disturbed (Fig. 7E and F) similar to that seen with the FKRP morphant alone.


Zebrafish models for human FKRP muscular dystrophies.

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

The gaps between myofibers can be repaired by co-injection of normal FKRP mRNA with MO2 but not all human mutant alleles. Co-immunostaining of 4 dpf fish co-injected FKRP mRNA with anti MHC (green) and anti laminin (red). (A) Control MO2. (B) Human normal FKRP mRNA co-injected fish. (C) FKRP MO2 injected fish. (D) Human mutated FKRP mRNA (L276I) co-injected fish. (E) Human mutated FKRP mRNA (C318Y) co-injected fish. (F) Human mutated FKRP mRNA (A455D) co-injected fish. Bar: 50 µm. In control and human control mRNA co-injected fish, the myofibers are adjacent at myosepta (arrows in A and B); however, FKRP morphant has the gap between myosepta (arrowhead in C). In L276I mutant fish, the gap is also narrowed (arrow in D). In C318Y and A455D mutant fish, there still remain gaps similar to that found in FKRP morphant fish (arrows in E and F).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

DDP528F7: The gaps between myofibers can be repaired by co-injection of normal FKRP mRNA with MO2 but not all human mutant alleles. Co-immunostaining of 4 dpf fish co-injected FKRP mRNA with anti MHC (green) and anti laminin (red). (A) Control MO2. (B) Human normal FKRP mRNA co-injected fish. (C) FKRP MO2 injected fish. (D) Human mutated FKRP mRNA (L276I) co-injected fish. (E) Human mutated FKRP mRNA (C318Y) co-injected fish. (F) Human mutated FKRP mRNA (A455D) co-injected fish. Bar: 50 µm. In control and human control mRNA co-injected fish, the myofibers are adjacent at myosepta (arrows in A and B); however, FKRP morphant has the gap between myosepta (arrowhead in C). In L276I mutant fish, the gap is also narrowed (arrow in D). In C318Y and A455D mutant fish, there still remain gaps similar to that found in FKRP morphant fish (arrows in E and F).
Mentions: In FKRP morphant embryos, the distance between the myosepta was reduced and the gap between myofibers was greater. To examine the nature of the protein structure at the gap, myofibers in mutant fish were examined by immunostaining with anti-MHC and anti-laminin. In control and human normal FKRP mRNA co-injected fish, the myofibers were adjacent at myosepta (Fig. 7A and B). The gaps between myofibers that were found in FKRP morphants (Fig. 7C) were also reduced with the injection of normal human FKRP mRNA (Fig. 7B). The length of myofibers was of similar size to that seen in control (Fig. 7A). Using the L276I mutant allele, the gap was reduced compared with FKRP morphants alone and the other two mutant alleles, C318Y and A455D (Fig. 7D). In C318Y and A455D mutant alleles, the gap was still wide and myofibers were disturbed (Fig. 7E and F) similar to that seen with the FKRP morphant alone.

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