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various intermediate filaments), intracellular signaling molecules (Gap-43,
myosin light chain kinase, PKC isoforms), and membrane components
(Reggie-1 and -2) (
Bastmeyer, Schlosshauer, & Stuermer, 1990; Becker
et al., 1998; Herdegen et al., 1993; Hieber, Agranoff, & Goldman, 1992;
Hieber, Dai, Foreman, & Goldman, 1998; Liu et al., 2002; Munderloh
et al., 2009; Perry, Burmeister, & Grafstein, 1987; Veldman, Bemben,
Thompson, & Goldman, 2007
). In addition to these effector molecules,
the expression of transcription factors including Atf3, Klf6, Klf7, Sox11,
and c-Jun also correlates with successful regeneration in RGCs of teleost
fish (
Veldman et al., 2007
). In some cases, functional data have
substantiated a role in successful axon growth. In supraspinal axons of
zebrafish, knockdown of the adhesion molecule L1 reduced by 70% the
number of regenerating axons. In RGCs, knockdown of Reggie-1 and -2,
which affect intracellular signaling by organizing plasma membrane
microdomains, caused a 60% reduction in the number of regenerating
RGCs (
Becker et al., 2004; Munderloh et al., 2009
). Knockdown of
alpha-1-tubulin reduced by nearly 80% the number of axons regenerating
from zebrafish retinal explants (
Veldman, Bemben, & Goldman, 2010;
Veldman et al., 2007
).
3.2. Gene profiling in zebrafish
Two groups recently used microarray analysis to examine the temporal pro-
file of gene expression in whole zebrafish retina after optic nerve crush
(
McCurley & Callard, 2010; Saul, Koke, & Garcia, 2010
). The use of
whole retina, however, makes it difficult to detect RGC-specific
changes. An elegant study by Veldman et al. used laser capture
microdissection and microarray analysis to identify more than 300
upregulated and 29 downregulated genes in purified RGCs (
Veldman
et al., 2007
). Of 19 genes selected for validation, all but one were
confirmed to change in RGCs by qRT-PCR or
in situ
hybridization. Six
genes were functionally tested by morpholino-mediated knockdown in
retinal explants (Socs3a, Socsb, Sox11a, Sox11b, Klf6a, and Klf7a). No
single gene knockdown produced an effect but combined Klf6 and Klf7
knockdown strongly reduced axon length and number. Given Klf7's
activity in mammalian CNS development and regeneration (above), its
emergence in this zebrafish profiling study provides an important example
of conserved gene function and the utility of cross-species comparison to
detect genes that regulate axon growth.
