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Figure 5. Growth cone mRNA repertoire is developmentally regulated: transcript number and complexity increases in 24 h. A , Schematic showing LCM collection of young stage 24 and old stage 32 growth cones. B , mRNAs identified in stage 24 growth cones were manually classified into different functional categories using NCBI Gene tool. In total, 286 mRNAs were identified of which 171 transcripts have a known function. The pie chart represents the functional classification of these 171 mRNAs. C , Comparison of functional classes of mRNAs between old (Fig. 2 A) and young ( B ) growth cones shows that most categories of mRNAs are upregulated with age (upward arrow), except for transcripts involved in protein synthesis and translation, demonstrating the increasing complexity of the growth cone mRNA pool with development. D , Comparative profiling shows that young growth cones have far fewer mRNAs than old growth cones (286 vs 958) and that the degree of overlap is ∼80%. E , IPA identified pathways that are specifically upregulated in stage 32 old versus stage 24 young growth cones. F , EphB4 mRNA localization in stage 32 growth cones was confirmed by fluorescence in situ hybridization. G , The specificity of the fluorescence signal was calculated by the mean pixel (fluorescence) intensity/unit area of antisense signal compared with its corresponding sense signal (n = 3 for each group). Statistical analysis performed using the Kruskal–Wallis test. Scale bar, 5 μm. H , Q-PCR results for EphB4 mRNA in LCM-isolated RGC cell bodies of old (stage 40) and young (stage 32) retina indicating similar expression levels at both developmental stages (n = 4 for each stage). Image published in: Zivraj KH et al. (2010) Copyright © 2010. This image is reproduced with permission of the publisher and the copyright holder. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.
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