Caudal Fin Regeneration in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 305

plasmid DNA fused with a reporter has supported a role for sonic hedgehog
signaling in bone formation and patterning during fin regeneration (Quint
et al., 2002). Ectopic expression of promoter activity has recently been applied
to the larval zebrafish model. Embryos are microinjected with constructs bearing
a promoter of interest fused to a firefly luciferase reporter gene (Alcaraz-Perez
et al., 2008).

22.6.5 Chemical Genetics

Zebrafish embryos are well suited for high-throughput chemical screening. Embryos
can be loaded into 96-well plates and remain viable for 5 days without added
nutrients. As such, zebrafish are commonly used in a tiered toxicology screening
approach to identify classes of compounds that affect biomarkers of specific tissues'
and systems' function. Chemical genetic approaches have additionally been em-
ployed to elucidate the role of Wnt signaling in epimorphic regeneration (Mathew
et al., 2007, 2008b; Chen et al., 2009). Through the use of a diverse synthetic
chemical library, a new class of Wnt pathway inhibitors was revealed to negatively
impact fin regeneration. This class of compounds prevents destruction of Axin
proteins, thereby preventing liberation of b -catenin from the APC-Axin-GSK3 b

inhibitory complex and increasing -catenin degradation. We have reported that

inappropriate activation of aryl hydrocarbon receptor (AHR) signaling following
2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD) exposure impairs tissue regeneration
in larval and adult zebrafish (Zodrow and Tanguay, 2003; Mathew et al., 2006).
Comparative toxicogenomic analysis revealed misexpression of Wnt signaling
pathway genes in TCDD-exposed fin regenerates (Mathew et al., 2008a).
R-Spondin1, a potent activator of Wnt/

b

-catenin signaling, was highly expressed
in regenerating fin tissue of TCDD-exposed fish. In support of this, we have shown
that transient knockdown of R-Spondin1 or LRP5/6 by morpholino injection
blocked TCDD-induced inhibition of fin regeneration suggesting that TCDD
impairs regeneration through inappropriate activation of canonical Wnt signaling
(Mathew et al., 2008a). Together, these studies support the concept that canonical
Wnt signaling plays a primary role in epimorphic regeneration and, in addition,
highlight the effectiveness of chemical genetics in revealing novel molecular targets
involved in epimorphic tissue regeneration.
Our group conducted a large-scale screen of a 2000-member chemical library to
identify modulators of larval tissue regeneration (Mathew et al., 2007). We found that
five members of the glucocorticoid family similarly impaired fin regeneration
(Mathew et al., 2007). These data were confirmed using a model GR agonist
beclomethasone dipropionate (BDP) (Fig. 22.4). Importantly, transient knockdown
of GR activity by morpholino injection blocked BDP-induced impairment of fin
regeneration (Mathew et al., 2007) (Fig. 22.4). These data indicate that the inhibitory
effects of BDP exposure on tissue regeneration are GR-dependent. These data further
support the relevance of using chemical genetics to unravel the signaling pathways
that dictate tissue regeneration.

b