Caudal Fin Regeneration in Zebrafish - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 304

Heat shock inducible transgenics expressing dominant negative gene products
have been extremely successful at unraveling the molecular signaling pathways that
dictate fin regeneration. Typically, heat shock promoters (Hsp) are used to control
expression of dominant negative gene products. This strategy is particularly useful
when examining the role of a gene that is required for development. Following
development, fish are heat shocked at 35-38 C to express the transgene fused to a
fluorescent reporter such as enhanced green fluorescent protein (EGFP). In the context
of fin regeneration, Hsp transgenics have supported a role for FGF signaling through
the overexpression of dominant negative Fgfr1 (Lee et al., 2005) and for Wnt- -

catenin signaling through overexpression of the negative regulator Dikkopf1, dom-
inant negative Tcf3, wnt8a, or wnt5b (Stoick-Cooper et al., 2007b).

b

22.6.3 Antisense-Mediated Gene Knockdown

Morpholinos are modified antisense oligos that knockdown gene function by
inhibiting mRNA splicing or translation initiation. Translation inhibition mor-
pholinos display sequence complementarity to the 5 0 untranslated region or
translation start site while splice site blockers have sequence complementarity
to intron-exon boundaries. Morpholino efficacy must be validated by immuno-
histochemistry or in situ hybridization dependent upon antibody availability and
researchers should be cognizant of potential off-target effects (Amsterdam and
Hopkins, 2006).
Morpholinos are widely used in fin regeneration studies in both adult and larval
models. The zebrafish larval fin regeneration model is particularly well suited for
efficient gene knockdown by morpholino injection. Morpholinos are injected into
either the yolk stream or cytoplasm at the one-cell stage resulting in transient
knockdown of gene expression. We have reported efficient gene knockdown in the
larval fin regeneration model (Mathew et al., 2007, 2008a, 2008b). It is important to
note that as embryogenesis proceeds, the morpholino concentration is exponentially
reduced. While morpholino-mediated gene knockdown is possible in adult fins, it
requires numerous injections per fish followed by electroporation to enable morpho-
linos to gain intracellular access. This is a result of more complex fin architecture
present in adult fins (18 lepidotrichia and 18 blastemas following amputation). In
addition, temporal restraints provide a significant limitation of morpholino injections
in the adult fin model. Following amputation, researchers typically wait 24 h before

microinjection and electroporation to allow thewound to heal rendering this approach
less well suited for the discovery of the signaling events that initiate blastema
formation (Thummel et al., 2006; Thatcher et al., 2008).

22.6.4 Ectopic Gene Expression and Promoter
Analysis

Ectopic expression of gene products suspected to impact regeneration has been
used to investigate regenerative mechanisms. In the larval model, injection of