Molecular Control of Axon Growth: Insights from Comparative Gene Profiling and High-Throughput Screening - International Review of Neurobiology - page 60

In the same way, HCS of axon growth is in the early stages of develop-
ment and clearly possesses untapped potential. Neurite outgrowth is
extremely sensitive to small changes in cell preparation and culture condi-
tions, and thus developing workflows to control variability and creating data
analysis methods to discern biological signal are important goals ( Buchser,
Slepak, Gutierrez-Arenas, Bixby, & Lemmon, 2010 ). Increasing throughput
is a second major goal, in particular to realize the potential of HTS to explore
combinatorial gene manipulations. To the extent that the enhancement of
axon growth is likely to depend on multiple pathways and cellular processes,
large-scale screens of gene combinations are most likely to uncover poten-
tially synergistic effects. A third improvement will involve enriching the data
collected in neurite outgrowth screens to make them more fully “high con-
tent.” One could conceive a screen in which, for instance, multiple neurite
outgrowth parameters were measured for each cell (number, length,
branching, etc.), along with a fluorescence-based indicator of metabolic
activity (phospho-S6 immunoreactivity, for instance), and perhaps the nu-
clear localization state of a transcription factor or signaling molecule of in-
terest. Obtaining these multiple readouts as genes are overexpressed or
knocked down in neurons will enable a much more detailed understanding
of gene function. In summary, as variability is brought under control,
throughput is increased, and multiparametric measurements are adopted,
HTS/HCA will likely be increasingly useful in identifying novel genes as
well as novel gene interactions that control specific aspects of the complex
biology of axonal regeneration.
ACKNOWLEDGMENTS
I wish to thank Drs. John Bixby, Vance Lemmon, Kevin Park, and Jae Lee for critical review
of this chapter.
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