Fig. 6

Gene expression changes induced by transcription factor (TF) over-expression, or by axotomy, quantified by RNASeq on laser dissected large-diameter dorsal root ganglion (DRG) neurons. (A) Principle component analysis. The first principal component (PC1) appears to reflect changes induced by AAV vector injection and transduction, while the second component (PC2) appears to primarily reflect changes induced by axotomy (i.e. the RAG program). AAV-injection itself does not appear to induce changes in PC2 but TF overexpression does in several groups, suggesting TF expression causes some induction of axotomy-like changes in gene expression. (B) Numbers of differentially expressed genes induced by each TF overexpression group. (C) Expression of a panel of RAGs following axotomy. With the exception of KLF6, all RAGs were induced by axotomy, showing that RNASeq on laser dissected neurons has good sensitivity. * False Discovery Rate < 0.05. (D) Expression of endogenous ATF3, KLF7 and MEF2 and viral-vector-expressed TFs and fluorescent marker genes in nerve injury and TF-overexpression groups. TF and fluorophore expression was detected as expected in all TF over-expressing groups. Note that endogenous and transgene ATF3 have identical coding sequences and so are not well distinguished. Similarly GFP and YFP also have almost indistinguishable sequences. (E) Clustered heat map of gene expression log fold-changes for RAGs in DRG neurons overexpressing TFs and days after sciatic nerve injury (SNI). TF-overexpressing groups cluster together in a separate tree to the SNI groups. In the TF groups ATF3 and ATF3/ KLF7/ MEF2 are the most similar to each other, while MEF2 is the least similar to the others. The majority of RAGs display broadly similar patterns of gene expression changes between the SNI at 1 and 7 days and the TF groups, outlined in boxes 1, 2, 5 and 6, with genes being up-regulated in 1 and 2 and down-regulated in 5 and 6. Boxes 3 and 4 contain the smaller group of genes where the SNI groups have opposite expression patterns to the TF groups. Key to log fold changes applies to B, C, D and E