Hepatotoxicity Testing in Larval Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 87

8.3 HEPATIC GENE KNOCKDOWN AND MUTATION
Genes identified in the zebrafish model are well conserved among other vertebrates
and as a result a plethora of zebrafish mutants have been identified in order to help
understand their homologs in mammals and humans (Haffter et al., 1996). N-Ethyl-
N-nitrosurea (ENU) has been a common mutagen used in zebrafish studies and has led
to hundreds of mutations in genes essential for numerous processes including pattern
formation, morphogenesis, organogenesis, and differentiation. Morpholino oligonu-
cleotides (MOs) (Kemper et al., 2003) and various mutant zebrafish studies have also
been key in allowing scientists to rapidly advance our knowledge of the functional
definition of genes required for normal development of the liver.
When injected into 1-4-cell stage zebrafish embryos, morpholinos have been
shown to successfully knock down gene expression by binding to and blocking
translation of specific mRNA (Nasevicius and Ekker, 2001). MOs are antisense
nucleic acid analogs that have ribosides converted to morpholines (C 4 H 9 NO) and
a phosphorodiamidate intersubunit linkage instead of phosphorodiester linkage
(Summerton and Weller, 1997). By injecting complementary sequences of hhex,
these MOs cause a significant reduction of liver size by 50hpf (Wallace et al., 2001),
indicating that hhex may play an important role in liver formation. Similarly, loss of
Hhex function in mutant mouse studies has shown that it impedes growth and
differentiation of an initially established liver diverticulum (Keng et al., 2000;
Martinez Barbera et al., 2000). In addition, the pescadillo (pes) gene, initially
identified in an insertional mutagenesis screen in zebrafish, appears to play an
important role during the growth phase (Allende et al., 1996). pes mutants exhibit
a smaller liver at 120hpf, although there is no obvious difference towild-type larvae at
72hpf. This suggests liver development becomes arrested during this time and
supports the conclusion that expression of pes precedes phases of active proliferation
in the liver and gut (Allende et al., 1996).
As well as being useful to identify genes involved in development and detox-
ification, mutants are often useful models for studying human diseases (van
Heyningen, 1997; Zon, 1999; Barut and Zon, 2000; Dooley and Zon, 2000; Amatruda
et al., 2002). For example, mutations on genes including lumpazi, gammler, and tramp
lead to liver necrosis (Chen et al., 1996) and the beefeater mutation exhibits liver
necrosis and impaired glycogen utilization, as seen in human glycogen storage
diseases (Pack et al., 1996). Sadler et al. (2005) also identified three mutants that
had phenotypes resembling different liver diseases. One mutant of a novel gene
named foie gras developed large, lipid-filled hepatocytes and therefore resembled
humans with fatty liver disease. Hepatomegaly and vesicle-filled hepatocytes were
observed in the second mutant that lacked vps18, a class C vacuolar protein sorting
gene. This also exhibited defects in the bile canaliculi and had marked biliary paucity,
indicating that this gene functioned to traffic vesicles to the hepatocyte apical
membrane and also could be involved in the development of the intrahepatic biliary
tree. This phenotype is comparable to that reported for individuals with arthrogry-
posis-renal dysfunction-cholestasis (ARC), a syndrome attributed to mutation of