Molecular Control of Axon Growth: Insights from Comparative Gene Profiling and High-Throughput Screening - International Review of Neurobiology - page 47

3.3. Screening in nonmammalian species
Nonmammalian species offer the exciting possibility of using intact animals
in HTS for axon regeneration. In C. elegans , single axons labeled by Green
Fluorescent Protein (GFP) can be severed by laser axotomy, and this tech-
nique was recently used to test 654 mutant lines for inhibition or potenti-
ation of axon regeneration ( Chen et al., 2011; Yanik et al., 2004 ). A
combined microfluidics/laser axotomy system has also been developed
and used to rapidly test a library of about 100 compounds in whole
animals ( Samara et al., 2010 ). As an alternative to laser-induced axotomy,
another study took advantage of a b -spectrin mutant in which axons
continually break and regenerate, and performed a screen of siRNAs to
identify genes that disrupt this ongoing regeneration ( Hammarlund, Nix,
Hauth, Jorgensen, & Bastiani, 2009 ). Zebrafish may also be a viable
model for in vivo screens of axon regeneration. For instance, automated
imaging, microfluidics, and laser axotomy of EGFP-labeled axons have
been combined to allow rapid screening of compounds or mutant lines in
a zebrafish model of spinal axotomy ( Pardo-Martin et al., 2010 ).
C. elegans thus represents an established platform for in vivo screening of
regeneration-associated genes, and zebrafish appear poised to follow. Can
experiments conducted in these species identify genes that are relevant to
mammalian CNS axon regeneration? The answer hinges on the degree of
evolutionary conservation in the underlying molecular mechanisms of axon
growth. The available information suggests a remarkable degree of conser-
vation. For instance, in the zebrafish microarray study of Veldman et al.
(2007) , of the 36 genes most strongly upregulated in regenerating zebrafish
RGCs, 20 had previously been identified as upregulated in regenerating
mammalian peripheral neurons. Of the remainder, many have not been
tested in mammals, so further research could very well identify additional
overlap. At a transcriptional level, Atf3, c-Jun, Sox11, and KLFs have been
linked to regeneration in both zebrafish retina and mammalian system. As
discussed earlier, Klf6 and Klf7 are required for successful RGC regeneration
in zebrafish, and heightened Klf7 activity in mammalian CST neurons is suf-
ficient to promote axon regeneration, providing a strong example of a con-
served role for this family of transcription factors across species and cell type
( Blackmore, Wang, et al., 2012 ). Indeed, some degree of evolutionary con-
servation appears to extend even as far as C. elegans . Second messengers such
as Ca 2 þ and cAMP are important for both mammalian and C. elegans regen-
eration ( Cai et al., 2001; Ghosh-Roy, Wu, Goncharov, Jin, & Chisholm,
2010; Neumann, Bradke, Tessier-Lavigne, & Basbaum, 2002 ). Moreover,