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termed HCA, is well suited to neurite outgrowth assays and involves a trade-
off between reduced screening throughput and the enhanced ability to mea-
sure subpopulation parameters (i.e., axon growth from individual neurons)
(
Jain & Heutink, 2010
).
The technical challenges inherent in culturing and transfecting neurons
have slowed the spread of automated assays in the regeneration field, but two
recent studies have used the CST and RGC microarray datasets as the basis
for
in vitro
screening experiments. In the first, more than 100 genes that are
developmentally regulated in RGC neurons were overexpressed in embry-
onic neurons, and automated image analysis was used to measure neurite
lengths in transfected neurons (
Moore et al., 2009
). This screen identified
a transcription factor, Klf4, as a potent suppressor of neurite outgrowth,
and subsequent experiments confirmed Klf4's ability to reduce axon length
in RGCs and cortical neurons. Indeed, further experiments showed that
additional KLF transcription factors also affect neurite length, raising the
possibility that coordinated activity of the KLF family may play an
important role in controlling regenerative capacity in developing neurons
(
Mooreetal.,2009
). In the second screening experiment, nearly 500 genes
regulated in developing CST neurons were overexpressed in postnatal cor-
tical neurons using electroporation in 96-well format (
Blackmore, Moore,
et al., 2010
). Again, automated image analysis facilitated quantification of
neurite lengths. This screen identified a number of regulators of neurite
length, including cytoskeletal interacting proteins and mediators of
intracellular signaling (
Blackmore, Moore, et al., 2010
). Interestingly, this
screen also identified another member of the KLF family of transcription
factors, Klf6, as a positive regulator of neurite length (
Blackmore,
Moore, et al., 2010
).
One critical question is whether these screening experiments, con-
ducted in immature neurons
in vitro
, are truly effective in identifying genes
that are relevant to axon regeneration in the mature CNS
in vivo
. The KLF
family of transcription factors, which both screens identified as important
regulators of axon growth, provides an important affirmative example.
Knockout of Klf4, a growth-suppressive KLF, produced a significant en-
hancement of axon regeneration by RGCs following optic nerve crush
in adult mice, demonstrating a role for KLFs in the regeneration of mature
CNS neurons. Very recently, viral delivery of an engineered Klf7 mutant
(transcriptionally active and stabilized) proved effective in enhancing axon
regeneration by adult CST neurons (
Blackmore, Wang, et al., 2012
).
These data illustrate the ability of
in vitro
screening campaigns to identify
