Zebrafish Assays for Identifying Potential Muscular Dystrophy Drug Candidates - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 228

which are attached to the extracellular matrix of the transverse myoseptum. Second,
loss of dystroglycan and the DGC results in less pleiotropic phenotypes than those
observed in mice and dogs. Third, generation of dystroglycan knockdown zebrafish
by simple injection of gene specific reagents is straightforward. Fourth, assessment of
disease phenotypes and drug effects in zebrafish is faster and cheaper than assessment
in other models.
In zebrafish, somites, responsible for controlling movement, are primarily
comprised of myotomes that are a significant component of the body plan. Each
V-shaped myotome contains muscle cells and myoseptum, a connective tissue that
separates dorsal and ventral somites (horizontal myoseptum) from myotomes (ver-
tical myoseptum). The horizontal myoseptum is visible as a black line running
through the middle of myotomes (Fig. 18.7). The structure and function of myo-
septum are similar to the those of mammalian tendons. Occasionally, disrupted
myoseptum in the posterior section of the zebrafish body was also observed. These
defects often cause bent notochord and deformed posterior body plan.
Several muscular dystrophy phenotypes have been generated in zebrafish using
different methods. Sapje , a well-characterized dystrophin gene mutant (Bassett et al.,
2003; Bassett and Currie, 2004; Guyon et al., 2007), exhibits defective muscle
attachment that causes progressive muscle degeneration and cell death. We note that
utrophin, a dystrophin homolog that can substitute for dystrophin in early development,
is not present in zebrafish muscle fiber ends (Bassett and Currie, 2003). Therefore, the
sapje mutant and the dystrophin morphant exhibit severe phenotypes more similar to
dystrophin/utrophin double knockout mice (Grady et al., 1997; Janssen et al., 2005;
Guyon et al., 2007) than to mdx mice, which are only deficient in dystrophin.
18.1.4 Technologies for Gene Knockdown
in Zebrafish
Several methods for gene knockdown in zebrafish have been described including
antisense morpholino oligonucleotides (MOs), negatively charged peptide nucleic
acids (PNAs), ribozymes, and long and short dsRNAs including siRNAs (Skromne
and Prince, 2008). MOs have been widely used to block mRNA translation or splicing
in zebrafish and phenotypes have been shown to be similar to those observed in the
zebrafish sapje mutant (Nasevicius and Ekker, 2000). However, sequence-specific
“off-target” effects, which are mediated through a p53-dependent cell death pathway,
were recently discovered (Robu et al., 2007) and careful design of controls is required.
Other modified oligonucleotides, including PNAs, have been shown to function with
potency and specificity in zebrafish (Urtishak et al., 2003; Wickstrom et al., 2004) and
ribozymes for generating knockdown no tail zebrafish have also been assessed (Xie
et al., 1997; Pei et al., 2007); however, neither of these methods is widely used.
MOs have also been used to knockdown zebrafish dystrophin genes and MO
zebrafish exhibited bent or curved tails and were less active than controls (Guyon
et al., 2003). Dystrophin siRNA knockdown animals exhibited similar phenotypes
(Dodd et al., 2004). In addition, hooked tail and muscle necrosis were observed in